Nucleic acids encoding proteins for early liver development

ABSTRACT

Early developing stage-specific liver proteins and the genes coding for them that have been isolated and sequenced are provided, and these genes and proteins can be utilized to diagnose and/or treat a wide variety of liver disorders and other ailments. Included in the proteins identified and isolated in the present invention are the proteins known as elf 1-3, liyor-1 (145), pk, protein 106, and praja-1, along with the nucleic acid sequences coding for these and other proteins. Since the early developing liver proteins of the invention arise during embryogenesis when the liver and other organs are in transition from an undifferentiated state to a differentiated one, these proteins are involved in tissue differentiation and thus can be utilized in methods of diagnosing and treating a variety of liver diseases and other disorders including those relating to oncogenesis and tissue repair. Accordingly, the isolated early developing liver proteins in accordance with the present invention should have implications for diagnosis and treatment of a range of diseases from end stage cirrhosis to hepatocellular carcinoma and many other disease conditions.

FIELD OF THE INVENTION

This invention relates to proteins isolated during early liver development and the genes coding for them, and to methods for their use in diagnosing and treating liver disease and other disorders.

BACKGROUND OF THE INVENTION

In the United States and other countries, end stage liver disease due to infection, genetic defects or alcoholic consumption is a major cause of widespread morbidity and mortality, causing great potential hardship and economic loss to millions of people throughout the world. In addition, numerous other diseases, including biliary problems and blood disorders, are associated with disruptions in the many functions carried out by the liver, including iron transport, hepatocyte formation and hematopoiesis. In general, severe problems associated with a breakdown of liver function are practically untreatable, and require a liver transplant as the only cure. However, in light of the great disparity between the number of patients needing liver transplants and the number of donors, thousands upon thousands of people are denied this operation, and transplantation is at the present time not a practical approach to the problem.

At the same time, the precise nature of liver development and the role of early developing liver proteins has not been well understood. To date, no growth factors specific to the liver have been identified or isolated, and the precise molecular mechanisms behind hepatocyte (liver cell) formation remain to be elucidated. There thus has been a long felt need to identify and understand the changes in gene regulation and expression in the developing liver, including the determination as to which genes are switched on and off as a hepatocyte forms and a liver develops. Accordingly, isolating and identifying the genes and proteins which play critical roles in early liver development would be beneficial in understanding the effect of gene regulation and expression in the differentiating liver, and in diagnosing and treating many diseases states involving the liver and liver functions.

SUMMARY OF THE INVENTION

Accordingly, it is thus an object of the present invention to provide genes comprising the nucleic acid sequences encoding early liver developmental proteins, including the liver proteins known as elf 1-3, liyor-1 (145), pk, protein 106, and praja-1.

It is further an object to provide isolated and purified early developing liver proteins encoding by the above genes.

It is still further an object to use the early developing liver proteins of the present invention to provide liver-specific growth factors for application in diagnosis and treatment of liver disorders.

It is still further an object to provide methods of diagnosing and treating end stage liver disease using the early developing liver proteins of the present invention. It is even further an object to provide methods of diagnosing and treating other liver disorders and other diseases, including carcinoma, degenerative neurological disorders, anemia, and ataxia, using the early developing liver proteins of the present invention.

These and other objects are achieved by virtue of the present invention which provides genes coding for various proteins which are involved in the differentiation of the developing fetal liver, including the proteins known as elf 1-3, liyor-1 (145), pk, protein , praja-1, and a number of other stage-specific genes coding for early-developing liver proteins, and methods for their use in diagnosis and treatment of a variety of liver diseases and other disorders.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will now be described in detail with respect to preferred embodiments thereof, which are to be taken together with the accompanying drawings, wherein:

FIGS. 1A-1B represent the nucleic acid sequence encoding the liyor-1 (145) protein (SEQ ID NO:1 and SEQ ID NO:2) in accordance with the present invention.

FIGS. 2A-2E represent the nucleic acid sequence encoding the elf-1 protein (SEQ ID NO:3 and SEQ ID NO:4) in accordance with the present invention.

FIGS. 2F-2I represent the nucleic acid sequence encoding the elf-2 protein (SEQ ID NO:5) in accordance with the present invention.

FIG. 2J represents the nucleic acid sequence encoding the elf-3 protein (SEQ ID NO:6 and SEQ ID NO:7) in accordance with the present invention.

FIGS. 3A-3B represent the nucleic acid sequence encoding the praja-1 protein (SEQ ID NO:8 and SEQ ID NO:9) in accordance with the present invention.

FIG. 4A-4B represent the nucleic acid sequence encoding the pk protein (SEQ ID NO:10) in accordance with the present invention.

FIG. 5 represents the nucleic acid sequence encoding the 106 protein (SEQ ID NO:11) in accordance with the present invention.

FIGS. 6A-6B represent the nucleic acid sequence encoding gene 20 (SEQ ID NO:12) in accordance with the present invention.

FIG. 7 represents the nucleic acid sequence encoding gene 36 (SEQ ID NO:13) in accordance with the present invention.

FIG. 8 represents the nucleic acid sequence encoding gene 41 (SEQ ID NO:14) in accordance with the present invention.

FIG. 9 represents the nucleic acid sequence encoding gene 112 (SEQ ID NO:15) in accordance with the present invention.

FIG. 10 represents the nucleic acid sequence encoding gene 114 (SEQ ID NO:16) in accordance with the present invention.

FIG. 11 represents the nucleic acid sequence encoding gene 118 (SEQ ID NO:17) in accordance with the present invention.

FIG. 12 represents the nucleic acid sequence encoding gene 129 (SEQ ID NO:18) in accordance with the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In accordance with the present invention, early developing liver proteins and the genes coding for them have been isolated and sequenced, and these genes and proteins can be utilized to diagnose and/or treat a wide variety of liver disorders and other ailments. In general, the present invention arose from the investigation of liver formation during embryogenesis when the liver and other organs are in transition from an undifferentiated state to a differentiated one. This setting captures the phases of liver formation beginning with ordinary sets of endodermal cells. In addition, the early steps in tissue differentiation are closely related to the process of oncogenesis and tissue repair, and thus the isolated early developing liver proteins obtained in accordance with the present invention should have implications for diagnosis and treatment of a range of diseases from end stage cirrhosis to hepatocellular carcinoma and many other disease conditions.

In the identification and isolation of the liver proteins of the present invention which are useful in early hepatocyte formation, the first step that was taken was to "capture" and analyze gene expression at different stages of early liver formation, particularly at those stages that emerge in the range of about days 9 through 14.5 in the mouse. In this regard, four embryonic liver cDNA libraries were constructed, such as at days 10.0, 11.5, 12.5 and 14.5 post coitus, and after subtractive hybridization, isolation of a group of stage-specific, liver restricted clones were isolated. As will be set forth in more detail below, sequence analysis has revealed that these clones encode a series of early developing liver proteins, which are generally "stage specific", i.e., they appear only at specific stages of development and not other stages, including elf proteins 1-3, liyor-1 (145), pk, protein 106, proteins coded for by genes 20, 36, 41, 112, 114, 118 and 129, and praja-1, as will be described further herein.

The initial project to identify and isolate developing liver proteins had four main objectives: (1) to construct early embryonic liver libraries; (2) to screen and characterize these early embryonic liver libraries with a group of probes comprising known growth factors (IGF-I, IGF-II, and IGFBP-2) and transcriptional activators (C/EBP and LFB1), known to be expressed in the developing liver; (3) to carry out subtractive hybridization utilizing these cDNA libraries and analyze subsequent subtracted clones for stage specificity by southern blot hybridization, sequence, transcript size, abundance, and tissue distribution; and (4) to develop a functional assay for these subtracted genes using embryonic liver explant cultures.

With regard to the main objectives of the invention, it was decided to focus on the four stages of liver development, particularly around days e10, e11, e12 and e14 (embryonic days post coitus) in developing mice. These four stages are defined developmental time points representing phases of liver development from undifferentiated mesodermal/endodermal cells to a well developed and differentiated fetal liver. These stages have generally been categorized as follows: (1) at around e9-10, a change in cell polarity occurs; (2) at around e10.5-11, invasion and migration of endodermal cells into surrounding mesenchyme occurs; (3) at around e11.5-12, pseudolobule formation, cords of hepatocytes form together with early sinusoids; and (4) at around e12.5-e14.5, the liver is marked by hematopoietic foci and fully differentiated fetal hepatocytes. cDNA libraries representing these stages would therefore represent "captured" mRNA species expressed in greater abundance during critical time periods for hepatocyte formation, enabling their isolation and providing a method for analyzing the changing pattern of gene expression during liver development.

Another aspect of the present invention is the development of useful methods of diagnosis and treatment of liver disorders and other diseases made possible by the identification and isolation of the genes for early developing liver proteins of the invention and the expression of those genes. In accordance with the investigations made regarding these early developing liver proteins, it is clear that the different genes and proteins identified are important for different aspects of liver development and can thus be utilized in treatments of the appropriate disease. During embryogenesis, the liver generally develops from a foregut diverticulum, and comprises four main cell types: the first is the hepatocyte, or endodermal lineage; the second are biliary tree canalicular cells or bile duct cells, the third are hematopoietic cells, and the fourth are the Kupffer cell/Ito cells. As will be set forth below, of the early developing proteins isolated and obtained in accordance with the present invention, it is believed that the elf proteins are important for the formation of the biliary tree, as shown by antisense experiments; praja-1 appears to be important for iron transport and essential for hepatocyte formation as well as hematopoiesis; liyor-1 (145) and pk appear to be important in Ito cell formation and fibrosis.

Accordingly, in accordance with the invention, it is contemplated that elf proteins 1-3 will be useful in the treatment of disorders such as cholestasis, biliary stones, hepatic obstruction, stricture, primary biliary cirrhosis and primary sclerosing cholangitis. In addition, the proteins praja-1, liyor-1 (145) and pk will be useful in treating end stage liver disease, anhidrotic ectoderm dysplasia, hepatocellular carcinoma, as well as anemia, such as sideroblastic anemia, ataxia, e.g., spinocerebellar ataxia, degenerative neurological disorders, anhidrotic ectoderm dysplasia, and hemochromatosis.

Even further, it has also been discovered that the protein praja-1 has been identified in cancerous colon tissue, which normally does not produce this protein. Accordingly, it is contemplated that in accordance with the present invention, a method of detecting and diagnosing colon cancer is provided wherein colon cells or tissues are taken from a patient being tested, and these cells or tissues are screened to determine the presence or absence of the praja-1 protein. Identification of praja-1 in the colon cells or tissues will allow for a determination of whether the cells are cancerous since praja-1 will generally not be detectable in non-cancerous colon cells.

In the preparation of cDNA libraries in conjunction with this invention, it was necessary to utilize the four developmental stages discussed above in order to isolate key early developing liver proteins that affect the formation of hepatocytes and the liver. Although these studies were performed on mice, the relevance of the stages of liver formation to human development is shown in the following summary of these investigations:

(1) Day 10 post coitus (e10, 34-39 somites) (Human day 27):

In the mouse, the primary liver diverticulum appears during the 10th day of gestation. It develops from a foregut indentation in the endoderm which arises at e7, at the boundary between the embryonic and extraembryonic region, anterior to the developing heart rudiment. At this stage, although the cells are committed to the formation of fetal hepatocytes, they are still epithelial in nature and the liver diverticulum is not viable in the absence of the surrounding heart mesenchyme. As this is the earliest stage possible when hepatocytes are undifferentiated, it was considered to be of great importance: some cells are poised to differentiate into hematopoietic cells and others into hepatocytes. Accordingly, a day 10.0 library was constructed in lambda Unizap, and no prior group had ever constructed a cDNA liver library at this stage.

(2) Day 11.5 post coitus (e11.5. 40-44 somites) (Human day 32):

This stage is characterized by rapid growth of the liver. Soon after the formation of the hepatic bud, the endodermal cells proliferate, disrupting the membrane separating the epithelium from the septum transversum, with the epithelial cells migrating into the mesenchyme. The liver at e11.5 consists of broad hepatic cords separated by large sinusoids containing nucleated erythrocytes. Hematopoietic foci are found intermingled with the hepatic cords. A cDNA library was constructed in lambda gtlO and lambda Zap from embryonic livers obtained at this stage, since although cells are proliferating rapidly, they still have not attained a fully differentiated fetal state.

(3) Days 12.5-13.0 post coitus (e12.5, Human days 35-45) (Embryo size: 7-9mm):

This stage is easily recognized by early signs of finger development as well as by the anterior indentation of the footplate. At this stage, the liver is well developed, all lobes being clearly visible; it contains many megakaryocytes as well as cells with erythropoietic activity. A CDNA library at e12.5 was constructed in lambda gt10 and lambda Zap as this was the earliest stage where fully differentiated fetal hepatocytes are seen.

(4) Day 14.5 post coitus (e14.5, Human days 51-57) (Embryo size: 20-32mm):

At this stage, individual, separated forefoot fingers can be seen; hair follicles in the skin can be recognized and the umbilical hernia is very conspicuous. This stage represents a well differentiated fetal liver containing scattered blood-forming foci. A cDNA library of this stage was constructed in lambda Unizap in order to facilitate subtraction with the day 10 library which was also constructed in lambda Unizap (Stratagene).

(5) Adult mouse liver:

At birth, day 19, there is a major "switch" in the expression of a large number of genes. From now until the stage at which adult liver is formed, enzyme synthesis of the urea cycle and gluconeogenesis are upregulated. Adult liver is no longer a center for hemopoietic activity except in pathological situations and hepatocytes do not enter de-differentiated states, though the liver still has regenerative capacity as seen in partial hepatectomy.

In conjunction with each of these stages of development, RNA was recovered from each stage, and the quality of the RNA obtained following dissection was assessed by Northern blot analysis using mouse Beta Actin from the Chiba Cancer Center Research Institute, Chiba, Japan. Table 1 shows the RNA yields obtained. The CDNA library construction at days e11.5 and e12.5 of the embryonic liver was carried out by conventional techniques, and the libraries of the day e10.0 and adult mouse liver were obtained using the Stratagene Unizap cDNA library kit. RNA yields and cDNA library size fractionation are shown in Tables 1 and 2, below.

                  TABLE 1     ______________________________________     RNA yields obtained for each stage            NUMBER OF   TOTAL RNA    POLYA + RNA     STAGE  LIVERS      (RNA per embryo)                                     (% of total RNA)     ______________________________________     e10.0  608         63 mg (1.04 mg)                                     26   mg(4%)     e11.5  48          60 mg (6.7 mg)                                     16.8 mg(3%)     e12.5  28          N.D.         40   mg(approx)      e4.5. 20          N.D.         N.D     ______________________________________

                  TABLE 2     ______________________________________     Early embryonic liver cDNA libraries and adult mouse liver library                         SIZE AFTER                         AMPLIFICATION OF     LIBRARIES              INITIAL SIZE                         2X106 CLONES  VECTOR     ______________________________________     e10.0    6.1 × 106                         2.0 × 1010                                       Lambda     Unizap     e11.5    5.2 × 106                         1.6 × 1010                                       Lambda Zap              4.1 × 107                         2.0 × 1011                                       Lambda gt10     e12.5    8.0 × 106                         1.6 × 1011                                       Lambda Zap              1.6 × 107                         4.0 × 109                                       Lambda gt10     e14.5    7.0 × 106                         2.0 × 1011                                       Lambda     Unizap     Adult    5.0 × 106                         2.1 × 1010                                       Lambda     Unizap     ______________________________________

The cDNA inserts for e10.5, e11.5, e12.5, e14.5 post coitus stage mice and the adult mouse liver were size selected on a Biogel A150 column (>500 bp) prior to ligation to the vector. Actin frequencies for the libraries are given in Table 3.

Qualitative analysis of cDNA libraries utilizing known developmentally regulated cDNAs were carried out in order to establish developmental profiles of important "early" genes that are significant in development, and these libraries were then screened with a specific number of probes. The following probes were obtained and used for screening these libraries, including: Insulin like growth factor I (IGF I), obtained from Dr. Derek le Roith (NIH); Insulin like growth factor II (IGF II) and IGF II binding protein -2(BP- 2) both obtained from Dr. Matt Rechler of NIH; LFB 1 obtained from Drs. Monaci, Nicosia and Cortese of EMBL in Heidelberg, and the C/EBP probe from Dr. Darnell of the Rockefeller University in New York, N.Y.

                  TABLE 3     ______________________________________     Clone frequencies for day 10.0, 11.5 and 12.5 libraries     (Carried out on Duplicate Screens)     Positive cDNA clones per 100,000 poly A+ containing cDNA clones     Probe      e 10.0  e11.5      e 12.5                                         Adult     ______________________________________     IGF I      N.D.    Nil        Nil   N.D.     IGF II     3       0          4     0     BP 2       1       1          0     7     LFB I      Nil     Nil        1     N.D.     C/EBP      N.D.    2          5     N.D.     Beta Actin 120     130        246   270     ______________________________________      N.D. = Not Done

The data shows that IGF I was not detected in any of the embryonic libraries, while IGF II was detected in increasing clone frequency from e6.5 to 8.5 (8 at e6.5, 8 at e7.5 and 38 at e8.5--data not shown) and was also detected in the e10.0 and e12.5 libraries (3 at e10.0 and 4 at e12.5--see Table 3). IGF II was not detected in the adult liver library. Interestingly, BP2 clone frequencies are similar to IGF II in the early e6.5, e7.5 and e8.5 libraries (data not shown), but in the liver cDNA libraries the clone frequencies differed, for BP2 only one clone per 100,000 being detected at e10.0 and e11.5, while 7 were detected in the Adult Liver Library compared to the greater numbers for IGF 11. This implied that its temporal and spatial expression in the embryo and fetus is different from IGF 11 and this was subsequently confirmed by in situ studies. LFB I was detected in the e12.5 library, but at one clone per 100,000 screened, which implied that it is not present in mitogenic cells, but that its level was regulated and increased from birth onwards. C/EBP was not present in the e6.5, e7.5, e8.5 or e10.0 libraries (data not shown) but was suddenly detected at day e11.5 and e12.5 in low abundance (about 2 clones/100,000 at e11.5 and 5 at e12.5), implying that while it is expressed, its level also may be regulated, albeit downward, in embryonic stages. Lastly, Beta Actin was used as a reference: all seven libraries had similar Beta Actin frequencies from 120-300/100,000 clones which is considered representative of such embryonic libraries.

Next, identification of stage specific clones by subtractive methods was carried out, and two subtracted libraries were then constructed. Two rounds of subtraction were carried out, and the resulting subtracted libraries comprised 64 clones (e11.5-12.5), and 174 clones (e10.5-14.5). Further characterization of these clones was carried out as follows: (1) Southern hybridization; (2) sequencing; (3) Northern analysis; (4) Zoo blot analysis; and (5) In vitro translation of protein.

In the Southern blotting of these clones, thirty-four clones were shown to be stage specific and not containing mitochondrial, ribosomal and globin sequences. DNA sequencing of these thirty-four stage specific clones was carried out in order to identify clones bearing homology to known developmental genes (such as cell polarity genes, homeobox genes, etc.), and the first 400 base pairs of each clone were sequenced. A detailed analysis was then carried out with respect to some of the clones which form a part of the present invention, including liyor-1 (145), protein 106, pk, and praja-1, since these clones exhibit true stage specificity and appear to belong to a set of genes encoding signal transduction proteins, which are of great interest in development currently, due to studies demonstrating their importance in cell lineage. Other stage-specific proteins which are coded for by genes in accordance with the invention are discussed further below. Studies carried out with regard to proteins such as praja-l and elf, as well as other early developing liver proteins, have elucidated the sequence of these proteins, as will be set forth in more detail below.

As an example of the tests used to elucidate the developmental expression of these liver proteins, the protein liyor-1 (145) was tested to determine whether these transcripts are differentially expressed during development, specific for mesoderm or endoderm derived tissues, or are expressed in adult mouse and human organs. Accordingly, tissues from mid-gestational embryos were analyzed to determine the role of 145 in liver development. In these tests, tissues were dissected from day 11 onwards, as it was at this stage that discrete hepatic, cardiac and other tissues could be dissected with ease, with subsequent RNA isolated being of good quality.

RNA hybridization with liyor-1 (145) DNA in different mouse tissues was studied by using polyA RNA obtained at various developmental stages using a 32P-labeled 1.1 Kb insert representing protein 145. The specificity of the developmental changes in the steady state levels of 145 was evaluated by also measuring the relative levels of Actin. This revealed a 2.4 Kb transcript at high stringency washes. Scanning densitometry of the respective bands revealed that maximal expression of 145 occurred in liver and heart, less so in other tissues, but specifically on day 11 and in decreasing abundance at days 12.5 and 14.5 (when Northerns were developed 1-2 months later).

Further characterization of the distribution of protein 145 RNA in adult tissues and its conservation in evolution has involved RNA analysis of adult mouse and human tissues. The protein 145 hybridizes to adult liver, kidney and testis as a 2.4 Kb transcript in liver and kidney and a 2.6 Kb transcript in adult testis, in very low abundance: both blots were developed after being exposed to film for over a month at -70° C. Similar tests conducted with regard to the elf protein and the nucleic acid coding for it showed that elf DNA is generally conserved across many different species, including human, monkey, rat, mouse, dog, cow, chicken and yeast, and is represented in all species studied except rabbit.

Finally, in accordance with the invention, a functional assay was established for subtracted genes with the goal to establish mouse embryonic liver explant cultures in the laboratory, as this is usually considered the major hurdle for antisense experiments due to the need to dissect extremely small tissue sections at day 9.5 when the liver bud is 0.2 mm. In this regard, the interactions of the neighboring cardiac mesoderm and foregut endoderm were studied and the subsequent changes in cell type specific gene expression were characterized, particularly with respect to alpha-fetoprotein and albumin expression, and partially with respect to epithelial basement membrane components. Methods of culturing liver explants in accordance with the invention are described below. The results obtained in these tests have shown that when cultured in the complete absence of mesodermal derivatives, hepatic endoderm deteriorates rapidly. Only 2 out of 15 such liver explants survived. Hematoxylin and eosin staining showed a necrotic endoderm with no apparent signs of hepatic differentiation. When associated with the surrounding mesoderm particularly cardiac mesoderm (en bloc dissections), the endodermal cells had proliferated and invaded the mesodermal strands. Hepatocytes were seen to be organized in cords separated by sinusoids with pseudo-lobule formation. All 15 out of 15 cultures from en bloc dissections were completely viable. These studies confirm prior explant studies demonstrating the necessity of surrounding mesoderm for liver formation.

Accordingly, cDNA libraries have been constructed for the four main stages of liver development, e10, e11.5, e12.5, e14.5 and for adult liver in the mouse. These have been shown to be truly representative of their respective mRNA species, by meticulous analysis utilizing initial RNA blot analysis, size fractionation, quantitative, and qualitative analysis. Northern analysis confirmed the stage specificity, and restricted expression of their transcripts: for 145 this comprised a 1.35, and 2.37 Kb transcript restricted to midgestational brain and liver tissue, and adult mouse and human Northern blot analysis revealed 145 transcripts in extremely low abundance in liver, kidney, testis. Further tests with regard to protein 145 reveals its sequence identity of 53% (20 S.D.'s) to rat Phospholipase C-γ (PLC-γ), and amino acid alignment of conserved section of 145 to PLC-γ identifies a split pleckstrin homology (PH) domain. Protein 145 (liyor-1) bears 99% identity at the amino acid level to the PH domain at the amino terminus of PLC-γ. The PH domain is an area of 100 amino acids that has been found in a number of proteins including serine/threonine kinases, GTPase activating proteins, phospholipases and cytoskeletal proteins, and is thought to be involved in signal transduction. Nuclear magnetic resonance spectroscopy has revealed that the PH domain of P- fodrin is an electrostatically polarized molecule containing a pocket which may be involved in binding of a ligand. Of immense interest is the fact that this pocket is related to the peptidyl-prolyl-cis-trans-isomerase FKBP in which this pocket is involved in the binding of the macrocyclic compound FK506. Accordingly, it is contemplated that protein 145 may indeed bear a pocket for `natural` ligand similar to FK506 and thus appears to be a potential factor for hepatocyte differentiation.

PLC-γ is regulated by a combination of SH2- domain dependent complex formation with tyrosine phosphorylated receptor tyrosine kinases, and its subsequent phosphorylation on tyrosine residues. An unique feature of PLC-γ and protein 145 is that both contain a split PH domain, which in the case of the PLC fills the gaps between the SH2-SH2-SH3 region and the surrounding X and Y catalytic domains. The SH2 domains mediate the high affinity interaction of PLC-γ with activated growth factor receptors such as epidermal growth factor (EGF) or platelet derived growth factor (PDGF) receptor. The PH domain similarly may be utilized as a specialized noncatalytic domain directing complex formation between protein kinases and their presumptive targets during liver development. In addition, the area of complete identity and split PH domain in 145 and PLC-γ is conserved in a number of other proteins through to TOR2, an essential yeast PI 3 kinase, and to v-abl. A parallel can be drawn to the SH2 domain: that proteins associating with activated growth factor receptors have quite distinct enzymatic properties, are structurally unrelated within their catalytic domains, yet contain a similar noncatalytic domain of approx 100 amino acids, called the src homology (SH) region 2. The SH2 domain was first identified in non receptor protein tyrosine kinase like Src and Fps, by its apparent ability to interact with the kinase domain and phosphorylated substrates. It is believed that during the evolution of cellular signaling mechanisms, the acquisition of SH2 domains conferred on PLC-γ and GAP the capacity to interact with transmembrane tyrosine kinases and therefore couple growth factor stimulation to PI turnover and the kinase pathway. PH domains are similarly conserved and may be utilized in the same way that SH2 domains are.

As indicated above, the protein liyor-1 (145) appears to be important in Ito cell formation and fibrosis, and is thus thought to be useful in treating end stage liver disease as well as other conditions including hepatocellular carcinoma, anemia, ataxia, and hemochromatosis. It is contemplated that the use of the protein Liyor-1 will be by administering to a suitable patient an amount of this liver protein effective to treat the specific condition of that patient, and this would be carried out using conventional means and regimens well known to one skilled in this art. The sequence of Liyor-1 which has been determined using the cDNA libraries of the present invention is shown in FIG. 1A and 1B (SEQ ID NOS:1 and 2), and suitable amounts of the liyor-1 (145) protein may be prepared in a conventional manner by expressing by recombinant or other means the nucleic acid coding for the 145 protein, after which the protein can be isolated and/or prepared into substantially pure form as needed. In addition, the 145 protein may be administered with any other suitable compound normally utilized for administration into a patient, such as a suitable pharmaceutically acceptable carrier.

As indicated hereinbelow in the examples, other genes for early developing liver proteins in accordance with the present invention have been isolated and sequenced, including the genes coding for the elf proteins, praja-1, pk protein, protein 106, and genes 20, 36, 41, 112, 114, 118 and 129. With regard to the elf proteins, these proteins were studied by analyzing mRNA from tissues from mid-gestation embryos. Tissues were dissected from day 11 onwards, as it was at this stage that discrete hepatic, cardiac and other tissues could be dissected with ease, and the subsequent RNA that was isolated was of good quality. RNA hybridization with elf DNA in different mouse tissues was studied by using polyA RNA obtained at various developmental stages using a ³² P-labeled 1.1 Kb insert representing elf. The specificity of the developmental changes in the steady state levels of elf was evaluated by also measuring the relative levels of Actin. This revealed a 2.4 Kb transcript at high stringency washes. Scanning densitometry of the respective bands revealed that maximal expression of elf occurred in liver and heart, less so in other tissues, but specifically on day 11, and in e12.5 and e14.5 in decreasing abundance (when Northerns were developed 1-2 months later).

In situ hybridization was then used to confirm elf expression in 11.5 heart and liver as well as to determine its expression pattern during earlier liver development, as will be set forth below in the Examples. The liver bud, which originates from foregut endodermal cells, grows into the septum transversum at the 9th day of gestation (13-20 somite stage). Between days 10.5 to 11.0 post coitus, a considerable degree of differentiation occurs: The liver enlarges substantially over this period, this increase in volume being due to the invasion of the mesenchyme of the septum transversum by the hepatic cords, and the initiation of hematopoietic activity in the liver. At day 9.5, a strong labeling of elf becomes apparent in the heart, and the pattern appears to be trabecular, including the wall of the cardiac anlage. A section of the sino-atrial chamber wall also shows a high intensity of elf expression. The surrounding tissue, particularly the caudal liver bud region does not show the presence of silver grains.

At the next stage, day 10.5, silver grains clearly highlight the developing liver, which appears as a horizontal structure (L) in this section. At this stage, the signalling is weakening in the developing heart tissue. The surrounding tissues are remarkable for the absence of silver grains. At day 11.5, a strong labeling of elf becomes apparent in the liver, which is larger in size. The heart at this stage only shows a weak signal posteriorly. As a control, in addition to sense probes, a riboprobe to alpha fetoprotein outlines the developing embryonic liver at days 11-12.

A comparison of the day 9.5 and 10.5 embryos, demonstrates a temporal and spatial expression of elf: the temporal gradient of a rise and fall of elf expression in the heart can be inferred from the strong staining in the developing heart at day 9.5 followed by a weaker staining at the next stage (day 10.5). Simultaneously, liver expression increases. The spatial gradient is apparent where silver grains increase in density on moving from the developing heart to the liver: at day 10.5, antisense RNA probes from elf cDNA hybridized specifically to day 9.5 cardiac mesenchymal tissue; expression at day 10.5 being restricted to cardiac and hepatic tissue, with elf expression finally being restricted to the liver in later 11.5 day embryos. Of note, elf expression was seen in embryonic livers at later stages (days 12.5, 14.5 p.c.), but only in decreasing abundance: the message being detected in these later stages when Northerns and in situs were developed a considerable time later. Sense probes to elf did not hybridize to any tissues. This indicates that ELF expression is not a sudden "on" "off" phenomenon, but more of a gradient pattern: consistent with the expression pattern of brain beta spectrin.

Alpha fetoprotein antisense RNA probes hybridized specifically to 11.5, 12.5, 14.5 embryonic mouse liver tissue, which is in agreement with previous studies of mRNA isolated from embryonic liver samples. The earliest stage that we were able to detect alpha-fetoprotein mRNA by in situ hybridization was at 10.5-11.0 days of gestation. Similar experiments with albumin mRNA have shown it to be expressed at day 9.5 in clusters of cells arising from foregut epithelium and in cords of cells seen to be invading the septum transversum. In experiments with alpha-fetoprotein, the liver was labeled at all subsequent stages (day 11 onwards), and, upon histological examination appeared to occur primarily in the endothelial cells. Hematopoietic cells appeared retractile but did not contain the hybridization grains that were visible over the alpha-fetoprotein positive cells. These experiments show that elf mRNA is localized to early embryonic heart, and then moving to e11 liver.

Next, it was determined that elf was a marker for the mesodermal component of liver formation. As Northern analysis had revealed elf expression to occur in day 11.5 heart and liver tissue, in situ localization was performed to investigate whether elf expression was restricted specifically to mesodermal tissue from the heart and the liver and was then compared to the endothelial expression of alpha fetoprotein. The main regions of mesoderm in the developing embryo are dorsal (somitic), intermediate, and lateral. Specifically, lateral plate mesoderm comprises somatic tissues (pleura, pericardium, peritoneum and limb bud), and splanchnic tissues (heart, epicardium, myocardium, connective tissue and smooth muscles of viscera and blood vessels, hemangioblastic tissue, adrenal cortex and spleen). The developing heart, at day 9 (13-20 somites), appears to be only region within the embryo where the endothelial elements of the circulation are surrounded by a vessel wall. The walls of the common ventricular and atrial chambers show an increasing degree of trabeculation. The space between the endothelial and myocardial elements is filled with loose mesenchyme called cardiac jelly. In situ hybridization of days 9 and 10 embryonic heart tissue using elf antisense riboprobes showed high levels of labeling to both the atrial and ventricular regions, highlighting the trabeculation.

Hepatic mesenchyme also originates from lateral plate mesoderm. The septum transversum part of the hepatic mesenchyme originates from the splanchnic mesoderm of the precardiac area and this is considered to be responsible for the subsequent differentiation of hepatocytes. However, tissue explant experiments have demonstrated that all derivatives of the lateral plate can replace hepatic mesenchyme for these later events. The initial experiments have shown that migrating endoderm must interact with mesenchyme for the former to differentiate into hepatocytes and recent studies investigating albumin mRNA expression, an indicator of hepatocyte differentiation, have confirmed these features; Initial expression of albumin mRNA occurs during the invasion of the septum transversum, when foregut endodermal cells clearly contact cardiac mesenchymal tissue. Similarly, primer extension analysis of albumin transcription has shown that the start site of transcription to occur at day 10.5 with a 15-20 fold increase in albumin mRNA upon liver organ formation by day 12.5. In our experiments using alpha fetoprotein as a marker for differentiated hepatocytes, it was obvious that while alpha fetoprotein expression is restricted to the later endodermal component of liver development, elf expression seems to occur in the loosely organized, lighter staining mesenchymal cells, initially cardiac mesenchyme (at day 9.5), then in both cardiac and hepatic tissue (at day 10.5) and then restricted to liver tissue (day 11.5 onwards); elf expression then decreases in abundance upon full embryonic liver formation. Examination of later histological sections (days 11 onwards) demonstrated a diffuse distribution of grains, and the hybridization signal with elf appeared to be localized in the perisinusoidal cells, but not in the hepatocytes.

That elf is expressed in early cardiac mesoderm, with subsequent expression being limited to hepatic mesoderm, indicates that this is a novel marker for the mesodermal component of liver development. Molecular markers have been invaluable in the dissection of inductive events in embryological studies. For instance, in Xenopus, vg-1, a member of the TGF-Beta family, now considered to be the strongest candidate for dorsal mesoderm induction, was in fact originally isolated by differential screening of mRNAs localized in the vegetal hemisphere of developing Xenopus eggs. Activins and other genes belonging to the TGF-Beta family such as vg-1, as well as wnt and BFGF families, represent components of the cascade leading to the commitment to particular mesodermal fate and all are strong candidates as mesoderm-inducing factors. Yet of these, only vg-1 has been demonstrated to be localized to the vegetal cells, the blastomeres responsible for mesoderm induction in vivo. Specific localization of vg-1 was vital and responsible for the persistence required in investigating its role as the inductive agent in mesoderm formation. Similarly, in isolating putative inductive agents required for liver formation, a key step is the localization of a new mRNA isolated from the embryonic livers. Accordingly, it is contemplated that elf and its associated regulatory genes will be of enormous potential benefit as a liver growth factor.

Further characterization of elf has involved RNA analysis of adult mouse and human tissues, and it was determined that elf hybridizes to adult liver, kidney and testis as a 2.4 Kb transcript in liver and kidney and a 2.6 Kb transcript in adult testis, in very low abundance: both blots were developed after being exposed to film for over a month at -70° C. Genomic DNA analysis of elf expression in DNA (genomic) from human, monkey, rat, mouse, dog, cow, rabbit, chicken and yeast indicates that elf is conserved across the species, being represented in all except rabbit DNA.

In vitro transcription and translation of elf, the latter using nuclease-treated rabbit reticulocyte lysate (promega) has revealed a 34 Kd protein, which is as predicted by the elf insert size and indicating that this insert is in frame for the coding sequence for a specific protein. These studies have established the principle that specific mesodermal mRNAs are localized in a way that guarantees their subsequent segregation to specific mesodermal tissue, in this case the presumed mesodermal component of the liver as shown by embryonic explant studies.

The elf protein has been sequenced, and it has been determined that at least three specific elf protein genes can be identified during early liver development. The sequences for these genes, known as elf-1, elf-2, and elf-3, are shown in the FIGS. 2A-2E (SEQ ID NOS:3 and 4), 2F-2I (SEQ ID NO:5) and 2J (SEQ ID NOS:6 and 7), respectively. As indicated above, it appears that the elf proteins 1-3 are probably important for the formation of the biliary tree during early liver development. Accordingly, it is contemplated that in accordance with the present invention, the elf proteins will be useful in treating various disorders associated with liver function, including cholestasis, biliary stones, obstruction, stricture, primary biliary cirrhosis, and primary sclerosing cholangitis. As would be readily apparent to one skilled in the art, methods of treatment using the elf proteins would comprise administration of an amount of an isolated elf protein that is effective to treat the specific disease condition described above. As also would be apparent, the elf proteins themselves can be prepared in a number of suitable ways by expression from the nucleic acid sequences indicated at FIGS. 2A-2J, including recombinant methods of producing these proteins, followed by separation, isolation and/or substantially purifying the elf proteins. The elf proteins once obtained in this manner can be put into any suitable form that is acceptable for use with patients. In addition, any of these three elf proteins may be administered with any other suitable compound normally utilized for administration into a patient, such as a suitable pharmaceutically acceptable carrier.

Another protein that has been identified and isolated in accordance with the present invention and which is contemplated to be used in a variety of therapeutic methods is known as praja-1. Praja-1 has now been studied in conjunction with the examination of early developing liver proteins, and an analysis of the amino acid translation revealed the presence of a COOH-terminal RING-H2 motif, which is a zinc finger variant. Additionally, Northern blot analysis of RNA from adult mouse showed expression of 3.1, 2.6, and 2.1 kb transcripts in liver, brain, and kidney, and an additional 2.3 kb transcript in testis. Expression of praja-I is also apparent in a colon cancer cell line, SW 480, and as set forth below, it is also contemplated that the praja-1 protein will be a useful marker in early detection of colon cancer.

It has also been learned that praja-1 maps to chromosome X, at about the 36 cM position. Other genes mapping to this general region include moesin (Msn), androgen receptor (Ar), interleukin-2 receptor gamma (IL-2rg), X-linked zinc finger protein (Zfx), and tabby (Ta). The syntony and conserved gene order between mouse and human X chromosomes allows comparison with human disease genes in the region. Human diseases in this region with mesodermal involvement include anhidrotic ectoderm dysplasia (eda) and sideroblastic anemia with spinocerebellar ataxia (asat), and it is thus contemplated that in accordance with the present invention, praja-1 will be useful in treating these disease conditions, as well as degenerative neurological disorders.

In vitro expression of praja-1 has shown that the translational product, which ran as two closely spaced bands of Mr=55.6 and 56.9 kD, is larger than the predicted ORF size of 47.4 kD. One possible explanation is that the expression product is very acidic, and acidic proteins such as granins are known to give anomalously high Mr on SDS-PAGE. The presence of two products suggests translation initiation at a second, internal ATG codon, such as at Met-19.

In addition, antisense studies to praja-I demonstrated that praja-I is essential for liver architecture formation. Preliminary antisense studies were performed at 1.25, 2.5 and 5 mfn concentrations, utilizing two different ODNs to praja-1. In these tests, liver and block explants were treated with these antisense ODNs compared with control (scrambled, sense or no ODNS). The results showed that control livers were generally larger than the antisense-treated livers, and control blocks showed early hepatocyte growth, cartilage growth, and very preserved bile ducts. Both livers and blocks treated with either antisense ODN to praja-1, showed minimal hepatocyte growth, cell necrosis, yet preservation of cartilaginous tissue, in a dose dependent manner.

In praja-1, aside from the RING-H2 finger, the stretch of thirty-four COOH-terminal amino acids just past this motif is especially rich in proline residues (17.6%); and, as stated, the protein in general is very acidic. Proline-rich domains are found in several mammalian transcription factors, such as that at the COOH-terminus of transcription factor CTF. Proline-rich regions and also acidic regions are likely to function in contacting other proteins. When considering the praja-I sequence as a whole, the rat Neurodapl gene has the highest similarity. Neurodapl is expressed abundantly in rat brain, with much smaller amounts in heart and skeletal muscle. Though praja-I likewise shows expression in brain, unlike Neurodapl (which is a larger 4.8 Kb transcript), it also expresses in liver and kidney. The subcellular localization of Neurodapl was shown to be concentrated around the endoplasmic reticulum (ER) and golgi of the cerebral cortex and facial nucleus, and especially in the postsynaptic density region of axosomatic synapses. Based on its subcellular localization, plus the presence of the RING-H2 finger, Neurodap I is probably linked to the secretory or protein sorting. This similarity to Neurodapl indicates that praja-I is most likely involved in protein-protein interactions, possibly in a protein sorting or secretary pathway involved during hepatocyte formation.

The gene coding the praja-l protein has been sequenced, and this nucleic acid sequence is depicted in FIGS. 3A-3B (SEQ ID NOS:8 and 9). As indicated above, it appears that the praja-1 protein is probably important for iron transport, and essential for hepatocyte formation as well as hematopoiesis. Accordingly, in accordance with the present invention, praja-1 can be used in methods of diagnosing and treating diseases such as end stage liver disease, iron storage disorders, hepatocellular carcinoma, as well as anemia, such as sideroblastic anemia, ataxia, such as spinocerebellar ataxia, and hemochromatosis. As would be recognized by one skilled in the art, these methods of treatment would involve administering of an effective amount of the praja-1 protein to the patient afflicted with one of the disease conditions set forth above. In addition, the isolation of the praja-1 protein could be obtained by expression of the nucleic acid sequence indicated at FIGS. 3A-3B which codes for the praja-1 protein, and this protein can be produced from its nucleic acid sequence in any suitable manner well known in the art such as recombinant means. Once isolated in this manner, the praja-1 protein can be obtained in a desired form, such as in substantially purified condition, and can be incorporated into any suitable mode of treatment that would be compatible with the patient in need of such treatment. In addition, the praja-1 protein may be administered with any other suitable compound normally utilized for administration into a patient, such as a suitable pharmaceutically acceptable carrier.

Even further, as indicated above, it has also been discovered that the protein praja-1 has been identified in cancerous colon tissue, such as in colon cancer cell line SW 480, which normally does not produce this protein. Accordingly, it is contemplated that in accordance with the present invention, a method of detecting and diagnosing colon cancer is provided wherein colon cells or tissues are taken from a patient being tested, and these cells or tissues are screened in any suitable manner which would identify the presence or absence of the praja-1 protein in the tested cells or tissues. In this manner, the identification of praja-1 in the colon cells or tissues from the patient will be indicative of a cancerous condition in the colon cells or tissues, and thus the present invention will provide a simple and effective method for determining at an early stage, when the disease is still in a treatable condition, if the patient appears to have contracted colon cancer. Conversely, the absence of praja-1 will generally be indicative of a non-cancerous state in the colon cells tested.

Still other genes coding for early developing liver proteins in accordance with the present invention have been identified and sequenced, and these proteins will also be useful in various methods of diagnosis and treatment of disease conditions associated with the liver or liver function. Included in these additional genes are those nucleic acids coding for a protein identified as pk, as depicted in FIGS. 4A-4B (SEQ ID NO:10), nucleic acids coding for a protein identified as protein 106, as shown in FIG. 5, and genes 20, 36, 41, 112, 114, 118 and 129, as shown in FIGS. 6-12. These proteins also appear to useful in hepatocyte formation and in treating liver diseases in a similar manner to many of the proteins discussed above, and in a manner similar to known growth factors should be useful in treating a variety of conditions. For example, protein pk appears to be important in Ito cell formation and fibrosis and thus appears to be useful in the same manner as protein liyor-1 (145). Accordingly, the protein pk, as prepared from the nucleic acid sequence indicated at FIGS. 4A-4B, will likely be useful in treating end-stage liver disease, hepatocellular carcinoma, as well as other disease conditions including anemia, ataxia, and hemochromatosis. As in the above cases, these early developing liver proteins may be administered with any other suitable compound normally used for administration to patients, such as suitable pharmaceutically acceptable carriers.

It is thus submitted that the foregoing embodiments are only illustrative of the claimed invention, and alternative embodiments well known or obvious to one skilled in the art not specifically set forth above also fall within the scope of the claims.

In addition, the following examples are presented as illustrative of the claimed invention, and are not deemed to be limiting of the scope of the invention, as defined by the claims appended hereto, in any manner.

EXAMPLE 1

In accordance with the cloning strategy of the present invention to identify genes involved in early mouse liver development, the gene Praja-1 has now been isolated, a gene with similar sequences to the Drosophila melanogaster gene goliath (gl), and which is involved in the fate of mesodermal cells ultimately forming gut musculatures, fat body, and the heart. Praja-1 is a 2.1 kb gene encoding a putative 423 amino acid ORF and includes a COOH-terminal RING-H2 domain. Using the Jackson Laboratory BSS panel, the praja-1 gene was localized on chromosome X at 36 cM, near the X inactivation center gene, Xist. Northern blot analysis demonstrated three transcripts (3.1, 2.6 and 2.1 kb) in mRNA from adult mouse tissues brain, liver, and kidney as well as in mRNA from developing mouse embryos (days 7, 11, 15 and 17 post coitus, or p.c.). In vitro transcription/translation yielded two products with a Mr of 55.6 and 56.9 kD. The presence of the RING-H2 domain, a proline-rich region at the COOH-end, and regions rich in acidic amino acids, leads to the hypothesis that the Praja-1 product is involved in mediating protein-protein interactions, possibly as part of a protein sorting or transport pathway. This is strengthened by the similarity of praja-l to rat Neurodap1, whose product has been shown to localize to the endoplasmic reticulum and golgi in brain.

The molecular mechanisms underlying hepatocyte differentiation are not well understood, and thus identifying the genes underlying the control of liver development will provide powerful tools for understanding liver function and development, and will allow the use of inducing liver differentiation for therapeutic purposes. As part of a strategy to clone such genes, a new RING-H2 finger gene, praja-1, was isolated. RING-H2 fingers, a type of zinc finger, are similar to RING fingers except that Cys4 is replaced by His (see Freemont, Ann. N.Y. Acad. Sci. 684:174-192 (1993); Lovering et al., P.N.A.S. 90:2112-2116 (1993)). Here it is shown that praja-1 possesses a RING-H2 motif near the COOH terminal. The RING-H2 motif is similar to that of the Drosophila melanogaster gl gene (Bouchard et al., Gene 125:205-209, 1993), and to the rat Neurodap1 gene (Nakayama et al., J. Neurosci. 15:5238-5248, 1995). Praja-1, which localizes to chromosome X, is expressed in mouse brain, liver, and kidney. The presence of the RING-H2 motif, plus the acidic, hydrophilic nature of the translation product, leads to the hypothesis that praja-1 plays a role in protein transport.

Materials and Methods

cDNA preparation and 3'-RACE PCR: RNA was isolated from livers of day 11 p.c. embryonic mice (ICR, Harlan Sprague-Dawley) using guanidine thiocyanate (Chomczynski et al., Ann. Biochem. 162:156-159, 1987). Poly(A)+ mRNA was isolated from total RNA using Dynabeads, as per manufacturer's instructions. First strand cDNA was made from poly(A)+ mRNA using the Promega Reverse Transcriptase System and the 3'-RACE primer 5'- (Frohman, In: M. A. Innis et al. (eds.), PCR protocols: a guide to methods and applications, Academic Press, San Diego, pp. 28-38., 1990). The 3'-RACE primer was also used as the reverse primer in the PCR reaction. The forward PCR primer, originally designed to amplify a conserved region of a clone 145/PH (pleckstrin homology) domain, was 5'. The PCR reaction mix contained cDNA from about 10 ng of poly(A)+ mRNA, 25 pmol of each primer, I mM DNTP mix, and 2.5 units of AmpliTaq DNA polymerase (Perkin-Elmer) all in 10 mM Tris, 1.5 mM MgCl₂, and 75 mM KCl, pH 9.2 in a final volume of 50 ml. The temperature program comprised 35 cycles of denaturation (94° C., I min), annealing (55° C., 1 min), and extension (72° C., 3 min), followed by an additional 8 minute extension. One of the resulting PCR products (CH7) comprised a 725 bp fragment, which was cloned into vector PCRII using the Invitrogen TA Cloning Kit for sequencing, and found by sequence analysis to possess a RING-H2 finger. The portion of the final cDNA clones which correspond to CH7 is indicated in FIGS. 3A-3B.

Library screening: The PCR product CH7 was labeled with a-32P!-dCTP (3000 Ci/mmol, Amersham) via primer extension using the reverse PCR primer plus AmpliTaq polymerase at 72° C. in PCR buffer (Konat et al., in PCR Technology: Current Innovations (H. G. Griffin and A. M. Griffin, Eds.), CRC Press, Boca Raton, pp. 37-42, 1994). The resulting antisense probe was used to screen plaque lifts of a whole embryonic mouse (day 11 p.c.) cDNA library in vector λZap (Stratagene). Positive plaques were picked and purified, and DNA was isolated from lysates using standard procedures (Silhavy et al., Experiments with gene fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1984). Inserts were excised from λZap DNA using EcoRI, and were subcloned into pGEM3Zf(-) (Promega) for sequencing and subsequent manipulations.

DNA sequence analysis: DNA sequence comparisons to existing sequences were performed utilizing BLAST searches in Genbank. Alignments were performed using the GCG program PILEUP.

Chromosomal mapping: Southern blot analysis of genomic DNA from C57BL/6J (B6) and Mus spretus (SPRET/Ei) using 32P!-labeled CH7 as a probe revealed a restriction fragment length polymorphism for the enzyme TaqI. This polymorphism was used to follow the inheritance of the praja-1 gene using the (B6×SPRET/Ei)×SPRET/Ei backcross panels (BSS) from The Jackson Laboratory Backcross DNA Panel Map Service (Rowe et al., Mammalian Genome 5:253-274, 1994). Linkage and order relative to other markers was determined by minimizing the number of multiple recombinants within each haplotype.

Northern blot analysis of Praia-l expression: Northern blots containing 2 micrograms of poly(A)+ mRNA from mouse tissues (Clontech) were probed with 32P!labeled CH7 antisense strand using Express Hyb hybridization solution (Clontech) at 68° C., washed according to manufacturer's instructions, and subjected to autoradiography. A 32P!-labeled b-actin probe supplied with the Northern blots was used as a control to normalize RNA levels in each lane.

In vitro transcription/translation:

A transcription/translation-coupled rabbit reticulocyte lysate system (Promega) was used, as per manufacturer's instructions for 35S!methionine labeling. Clones of praja-1 in pGEM3Zf(-) plus a luciferase control clone were used with T7-RNA polymerase (sense direction). Each reaction comprised 12.5 ml rabbit reticulocyte lysate, 1 ml reaction buffer, 0.5 ml 1 mM amino acid mix minus methionine, 0.5 ml T7-RNA polymerase, and 20 units RNasin, all in 25 ml final volume. After a 90 min incubation at 300° C., products were lysed in SDS/mercaptoethanol treatment buffer and separated on a 10% SDS-polyacrylamide gel according to Laemmli, Nature 227:680-685 (1970). Proteins were electroblotted onto a BAS-NC membrane (Schleicher & Schuell) using a BioRad Trans-Blot apparatus according to manufacturer's instructions. Labeled products were visualized by autoradiography.

Results:

Isolation and sequence analysis of the novel gene. praja-1:

As part of the analysis of genes involved in liver development and function, we amplified the 3' end of a previously undescribed gene, CH7. We used the CH7 probe to screen a mouse embryonic cDNA library and isolated two overlapping clones, praja-1-5 and praja-1-6. Sequence analysis of the consensus overlap region revealed an open reading frame (ORF) of 424 amino acids, with a predicted size of 47.4 kD. Hydropathy analysis (Kyte et al. J. Mol. Biol. 157:105-132, 1982; not shown) shows that the translation product is highly hydrophilic, with no hydrophobic leader or membrane-spanning regions. The translation is also very acidic, with a pI of 4.6 and containing 17.7% acidic residues (Asp plus Glu). The putative ATG start codon indicated in FIGS. 3A-3B was selected because it is the upstream-most ATG that is in-frame with the ORF, and is preceded 21 bp upstream by a TAG stop codon. The context of this ATG, however, is only a weak fit to the consensus Kozak recognition sequence GCCACCatgG in that it does not have a purine at -3 nor a G at +4 (reviewed by Kozak, Genome 7:563-574, 1996). Sequence analysis of the amino acid translation revealed the presence of a COOH-terminal RING-H2 motif, which is a zinc finger variant (Freemont, supra). those of several other RING-H2 containing proteins.

Linkage analysis places Praia-l on mouse chromosome X:

A restriction fragment length polymorphism for praja-1 was identified using CH7 as a probe on a Southern blot containing DNA from the two parental strains digested with several restriction enzymes (TaqI, BglII, EcoRI, EcoRV, HindIII, HincII, KpnI, PstI). For every enzyme used, C57B16/J had only a single restriction fragment, while two fragments were always observed within the SPRET/Ei lane. A polymorphism obtained using TaqI was used to type the inheritance of the C57B1/6J allele in the BSS panel. There are two Spretus bands S1 and S2 and one C57B1/6J band B1. After comparison of the praja-1 genotypes to other genes typed within the database, it was determined that praja-1 maps to mouse chromosome X at about the 36 cM position. The S1 band is the praja-1 allele on X chromosome of SPRET/Ei. The S2 TaqI fragment appears in every backcross animal. Since all males from the backcross contain this allele, it is not localized to the X chromosome. Since females also have the S2 band, it is not Y-linked. Therefore S2 is an autosomal locus that contains sequence homology to the praja-1 probe sequence. Other genes mapping to this general region include moesin (Msn), androgen receptor (Ar), interleukin-2 receptor gamma (Il2rg), X-linked zinc finger protein (Zfx), and tabby (Ta). This area is also 1.1 +/- 1.1 cM from the Xist locus. Further studies are needed to determine if praja-1 is not expressed on inactivated X-chromosomes and if it plays a role in X-inactivation. The syntony and conserved gene order between mouse and human X chromosomes (Herman et al., Genome 6:S317-S330, 1996) allows comparison with human disease genes in the region. Human diseases in this region with mesodermal involvement include anhidrotic ectoderm dysplasia (eda) and sideroblastic anemia with spinocerebellar ataxia (asat).

In vitro expression produces a protein product larger than the predicted size. An autoradiogram of the in vitro transcription/translation products of clones praja-1-5 and praja-1-6 showed that only praja-1-5 produced a significant product. The product, which ran as two closely spaced bands of Mr=55.6 and 56.9 kD, is larger than the predicted ORF size of 47.4 kD. One possible explanation is that the expression product is very acidic, and acidic proteins such as granins are known to give anomalously high Mr on SDS-PAGE (Huttner et al., Trends Biol. Sci. 16:27-30, 1991). The presence of two products suggests translation initiation at a second, internal ATG codon, such as at Met-19.

Praja-1 transcripts are present in embryonic and in mouse tissues. Northern blot analysis of RNA from adult mouse showed expression of 3.1, 2.6, and 2.1 kb transcripts in liver, brain, and kidney, and an additional 2.3 kb transcript in testis. The praja-1 protein is unlikely to be a membrane receptor, since it lacks a hydrophobic transmembrane domain. The uniform hydrophilicity suggests a soluble protein. The praja-1 RING-H2 motif is shown aligned with those from several other proteins. RING fingers are generally thought to function in protein-protein interactions (Borden et al., Curr. Opinion Struct. Biol. 6:395-401, 1996; Saurin et al., Trends Biochem. Sci. 96:208-214, 1996). To cite a specific example, if either of the two cysteines that comprise the Zn++ binding site of the RING finger of acute promyelocytic leukemia protooncoprotein PML are mutagenized, then the nuclear multiprotein complex, or so-called nuclear bodies, fail to occur (Borden et al., EMBO J. 14:1532-1541, 1995). The authors conclude that the PNM RING domain, and probably other RING finger domains, are involved in protein-protein interactions.

In praja-1, aside from the RING-H2 finger, the stretch of thirty-four COOH-terminal amino acids just past this motif is especially rich in proline residues (17.6%); and, as stated, the protein in general is very acidic. Proline-rich domains are found in several mammalian transcription factors, such as that at the COOH-terminus of transcription factor CTF, and proline-rich regions and also acidic regions are likely to function in contacting other proteins (Mitchell et al., Science 245:371-378, 1989). A BLAST search of the proline-rich COOH-terminus revealed no significant matches to any protein in the available databases, however, when considering the praja-1 sequence as a whole, the rat Neurodap1 gene has the highest similarity; the alignment is presented in FIG. 15.

Neurodap1 is expressed abundantly in rat brain, with much smaller amounts in heart and skeletal muscle. Though praja-1 likewise shows greatest expression in brain, unlike Neurodap1 it also expresses in liver and kidney. The subcellular localization of Neurodap1 was shown to be concentrated around the endoplasmic reticulum (ER) and golgi of the cerebral cortex and facial nucleus, and especially in the postsynaptic density region of axosomatic synapses (Nakayama et al., supra). Based on its subcellular localization, plus the presence of the RING-H2 finger, the authors concluded that Neurodap1 is probably linked to the secretory or protein sorting. Praja-1 does differ from Neurodapl in several respects, however. In addition to being expressed in some different tissues than Neurodap1, praja-1 encodes for a product that is smaller (47.4 kD, based on the composite of the clones in vs. 77.9 kD for Neurodap1. The difference in size is at the N-terminus of the proteins. The largest transcript we observed for praja-1 was 3.1 kb, whereas Neurodapl exists as a single 4.8 kb transcript on Northern blots of rat brain mRNA.

In light of the fact that BRCA1, which possesses a RING finger, has an acidic pI, and is a secretory protein, also has properties of the granin family of proteins (Jensen et al., Nature Genet. 12:303-308, 1996), we examined praja-1 for a granin signature. We found no region in the praja-1 translation that gave a perfect match to the consensus E N/S!LX A/D!X D/E!XEL, though two regions matched five of the seven conserved residues. We were also unable to demonstrate the presence of clear coiled-coils, which are present in BRCA1 and proteins with the previously-mentioned tripartite structures. In these respects, praja-1 is more similar to Neurodap1 than to proteins such as BRCA1. Also, though the RING-H2 finger in praja-1 shows much similarity to that from the D. melanogaster goliath (gl) protein, the goliath protein possesses an alkaline pI (8.9) and no sequence similarity to praja-1 outside of the RING-H2 finger. The RING-H2 motif plus acidic and proline-rich regions, and similarity to Neurodap1, leads to the conclusion that praja-1 is involved in protein-protein interactions, possibly in a protein sorting or secretory pathway.

EXAMPLE 2

In accordance with the present invention, investigations were made with regard to the induction of differentiation in liver tissues in order to isolate and identify early developing liver proteins for use in therapies involving the liver and liver functions. In the developing fetus, inductive interactions, intercellular communication and the establishment of cell polarity are critical for growth and patterning during development. However, the precise mechanisms by which these effect hepatocyte differentiation or liver development have not previously been elucidated. Mammalian liver development was first recognized to be established through a specific sequence of interactions between mesenchymal and endodermal embryonic tissues. At 9.5 days of mouse gestation, upon signaling from the cardiac mesenchyme, endodermal cells from the liver diverticulum proliferate and migrate into the surrounding septum transversum. This specific area of loose mesenchyme in turn differentiates into hepatic mesenchyme and a liver bud is finally recognizable microscopically at about 10.5 days of gestation. This hepatic mesenchyme is continually responsible for the hepatocyte proliferation which then proceeds throughout embryonic life (Le Douarin, Med. Biol. 53:427-455, 1975). Albumin transcription can be detected as early as at day 9.5 (Cascio et al., Development 113:217-225, 1991), implying that hepatocyte differentiation begins when hepatic endoderm comes into contact with cardiac mesoderm. As a first step towards the analyses of signal transduction pathways regulating such a restricted pattern of gene expression, molecular markers as well as regulatory genes are required to identify the interactions required for liver development.

The dissection of gene regulatory pathways in the liver has led to the identification and characterization of transcriptional activators, C/EBP, DBP, LFB 1/HNF 1, 3 and 4 (Johnson, Cell Growth Differ. 1:47-52, 1990; Kuo et al., Development 109:473-481, 1990; Frain et al., Cell 59:145-157, 1989), of liver specific genes, such as α-fetoprotein and albumin (Tilghman, Oxford Surveys on Eukaryotic Genes, Oxford University Press, 1985). Yet, with the exception of HNF4, 3 α and β (Ang et al. Development 119:1301-1315 (1991) and Cell 78:561-574, 1994), none of the above have been found to play a definitive role in determining cell-lineage and regional specification of the developing liver. The small volume of liver buds (approximately 4×10⁻² mm³) yields even smaller quantities of proteins, DNA and messenger RNA thus making the molecular analysis of liver development difficult. Therefore, the construction of early embryonic liver cDNA libraries, and performing subtractive hybridization still remains the most plausible and comprehensive method of obtaining an unbiased catalogue of genes required during early mouse liver development (see Harrison et al, Development 121:2479-2489, 1995).

The isolation of markers would provide further insight into identifying transcriptional activators and growth factors involved in such a restricted pattern of gene expression, and eventually provide an approach to identifying signal transduction pathways involved in hepatocyte differentiation. In some cases, these pathways have been characters as in patterning and axis formation of the vertebrate head and body (Oliver et al., Development 121:693-705 (1995); Kessel et al., Science 249:374-379, 1990). For example, in Xenopus, a network involving brachvury, activin and wnt-related genes, is responsible for mesoderm induction, somitogenesis, myogenic and sclerotomal differentiation (see, e.g., Wilkinson et al., Nature 343:657-659 (1990); Herrmann et al., Development 113:913-917 (1991); Green et al., Trends Genet. 7:245-250 (1991); Sokol et al., Cell 67:741-752 (1991) ; and Smith et al., Cell 67:753-767, 1991), and dorsal ventral axis formation results from Xgsk-3 (the Xenopus homologue of Drosophila zw3/shaggy) phosphorylating its Xenopus homologue of armadillo, β catenin thus regulating the level of β catenin available for dorsal axis formation. However, there are no available molecular markers nor pathways which characterize either earlier liver development, nor its crucial mesodermal component.

In accordance with the present invention, it has been possible to identify and characterize such molecular markers and possible inductive transcripts for liver development. As set forth below, the characterization of the elf protein is described, and the expression of this protein may mark the separate components of liver development. The "bottom up" approach with regard to this characterization in general has led to the identification of a totally unexpected group of genes, and in particular, this is described with regard to the elf protein, which is probably involved in playing a role in establishing cell polarity by interactions at the surface membrane.

Characterization of cDNA libraries:

The four stages in liver development (e10, e11, e12, and e14, where e=embryonic) are defined developmental time points from undifferentiated mesodermal/endodermal cells to a well developed and differentiated fetal liver. A change in cell polarity occurs at e9-10. At e10.5-11, invasion and migration of endodermal cells into surrounding mesenchyme occurs; at e11.5-12, pseudolobule formation, cords of hepatocytes form together with early sinusoids. cDNA libraries representing these stages would therefore, represent "captured" mRNA species expressed in greater abundance during crucial time periods for hepatocyte formation, enabling their isolation and providing a method for analyzing the changing pattern of gene expression during liver development.

Libraries containing 5.0×10⁶ -4.1×10⁷ independent clones were generated from the largest cDNA fractions. Current estimates demonstrate that a library containing 5.0×10⁵ clones (Sambrook et al, Molecular cloning, a laboratory manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989) is a representative library with a 99% probability that rare transcripts (less than ten copies per cell) are present. Our libraries are therefore likely to be truly representative of their respective mRNA species for that stage.

Qualitative and developmental profiles of the libraries:

These were obtained, utilizing genes, such as IGF-II, IGFBP-2, IGF1, C/EBP, HNF/LFBI known to be expressed at different time points in developing liver. The data in Table 2 below demonstrate that IGF-I was not detected in any of the embryonic libraries, while IGF-H was detected in the e10.0 and e12.5 libraries (3 at e10.0 and 4 at e 12.5). IGF-II was not detected in the adult liver library. Interestingly, BP-2 clone frequencies are similar to IGF-II in the early e6.5, e7.5 and e8.5 libraries (data not shown), but in the liver cDNA libraries the clone frequencies differed, for BP-2 only one clone per 100,000 being detected at elO.0 and ell.5, while 7 were detected in the adult liver cDNA library compared to the greater numbers for IGF-II. This implied that its temporal and spatial expression in the embryo and fetus is different from IGF-II and this was subsequently confirmed by in situ studies. HNF1/LFB I detected in the e12.5 library was suddenly detected at day 11.5 and 12.5 in low abundance (2 clones/100,000 at e11.5 and 5 at e12.5), confirming that while it is expressed, its level also may be regulated, albeit downward, in embryonic stages. Lastly, mouse β-Actin was used as a reference: all seven libraries had similar β-Actin frequencies from 120-300/100,000 clones which is considered representative of such embryonic libraries.

Identification of stage specific clones by subtractive methods:

Two subtracted libraries were then constructed as previously described, comprising 64 clones (e11.5-12.5), and 174 clones (e10.5-11.5). Further characterization of these clones was carried out by Southern hybridization, sequencing, Northern blot analysis, Zoo blot analysis, and in vitro fertilization of protein. Using Southern blotting, thirty-four clones were shown to be stage specific and not containing mitochondrial, ribosomal and globin sequences, and further analysis was carried out on elf.

Identification and developmental regulation of elf transcripts:

Elf mRNA in tissues from mid-gestational embryos were analyzed, and tissues were dissected from day 11 onwards since it was at this stage that discrete hepatic, cardiac and other tissues could be dissected with ease, and the subsequent RNA isolated was of good quality. Using a 32P-labeled 1.1 Kb insert representing elf, the specificity of the developmental changes in the steady state levels of elf was evaluated by also measuring the relative levels of β-Actin. This revealed a 2.1 Kb transcript at high stringency washes. Scanning densitometry of the respective bands revealed that maximal expression of elf occurred in liver and heart, less so in other tissues but specifically on day 11, and in 12.5, 14.5 in decreasing abundance (when Northerns were developed 1-2 months later).

Sequence analysis of elf:

After subtraction hybridization, one stage specific clone was analyzed in detail: sc32. The initial libraries were then screened at high stringency (0.2×SSC, 60°), to obtain overlapping clones for sc32. Positives were picked, and after in vivo excision (Stratagene) into Bluescript, these were sequenced using the dideoxy chain termination method using oligonucleotides corresponding to previously determined sequence. Of the seven clones picked, three were found to be overlapping to sc32 and included sequence encoding elf. Confirmation of the identity of the clones and elf was carried out by Northern blot analysis of mouse embryonic tissues. In the case of elf, this gave rise to the same initial 2.1 Kb transcript with sc32 as a probe. A start codon was not present suggesting that we had not cloned the 5' end of the cDNA. However, the northern blot showed a 2.1 Kb transcript, thus suggesting that we had cloned complete elf and this probably represented a spliced form of β-fodrin. The authenticity of the 3' end of the elf sequence was confirmed by the comparison of the elf sequence with the expressed sequence tags (EST) database. Although no mouse ESTs for elf sequence were found, three different human EST clones were found to span the region of unique last 100 nt and the 5' adjacent sequence, suggesting the existence of elf homolog in human cells.

Prior sequence analysis has shown elf to bear 80% identity to β-fodrin, a non erythroid β-spectrin. Our sequence to elf is located between domains II and III of the β-spectrins. Domain II comprises 17 repeats of a 106 amino-acid motif and an ankyrin binding domain. The ankyrin binding domain is required for the correct subcellular localization of adducin, ankyrin and the Na+,K+ ATPase, without which cell morphology is disrupted. Domain II comprises a C terminal domain which contains varying numbers of residues (52-265) in alternatively spliced forms giving rise to tissue specific expression (Hu et al., J. Biol. Chem. 267:18715-18722, 1992), as well as the PH domain.

In situ localization of elf:

In situ hybridization confirmed elf expression in 11.5 heart and liver and determined its expression pattern during earlier liver development, using elf sense probes and alpha fetoprotein antisense probes as controls. The hepatic diverticulum, which originates at the foregut-midgut junction, begins to grow into the septum transversum at the 9th day of gestation (13-20 somite stage). Between days 10.5 to 11.0 p.c., a considerable degree of differentiation is seen in this primitive liver. The liver enlarges substantially over this period: the increase in the overall volume being due to the invasion of the mesenchyme of the septum transversum by the hepatic cords, and the initiation of hematopoietic activity in this organ. At day 9.5, a strong labeling of elf becomes apparent in the cardiac silhouette: the pattern appears to be trabecular, including the wall of the cardiac anlage. A section of the cephalad chamber (sino-atrial chamber) wall also bears a high intensity of elf expression. The surrounding tissue, particularly the caudal liver bud region does not show the presence of silver grains. At the next stage, day 10.5, silver grains clearly highlight the developing liver, which appears as a horizontal oriented structure (L) in this section. At this stage, the signaling is weakening in the developing heart tissue. The surrounding tissues are remarkable for the absence of silver grains. At day 11.5, a strong labeling becomes apparent in the liver, which is larger in size. The heart shows an extremely weak signal: silver grains being visible in only a single streak posteriorly. At this stage, elf expression also appears in the umbilical cord. As a control, in addition to sense probes, a riboprobe to α-fetoprotein outlines the developing embryonic liver at day 11-12.

A comparison of the day 9.5 and 10.5 embryos demonstrates a clear temporal and spatial gradient of maximal tissue staining with silver grains representing elf riboprobe: the temporal gradient of a rise and fall of elf expression in the heart may be inferred from the strong staining in the developing heart at day 9.5 followed by a weaker staining at the next stage (day 10.5). Simultaneously, liver expression increases. The spatial gradient is apparent from the developed patterns of these tissues which showed that silver grains increase in density as one moves from the developing heart to the liver: at day 10.5, antisense RNA probes from elf cDNA hybridized specifically to 9.5 day cardiac mesenchymal tissue; expression at day 10.5 being restricted to cardiac and hepatic tissue; elf expression finally being restricted to the liver in later 11.5 day embryos. Of note, elf expression was seen in embryonic livers at later stages (days 12.5, 14.5 p.c.), but only in decreasing abundance: message being detected in these later stages when Northerns and in-situs were developed a considerable time later. Elf sense probes did not hybridize to any tissues.

Alpha fetoprotein antisense RNA probes hybridized specifically to 11.5, 12.5, 14.5 embryonic mouse liver tissue, in agreement with previous studies of mRNA isolated from embryonic liver samples (Tilghman et al., P.N.A.S. 79:5254-5257, 1982). The earliest stage of detection of α-fetoprotein mRNA by in situ hybridization was at 10.5-11.0 days of gestation. Similar experiments with albumin mRNA (Cascio et al., Development 113:217-225, 1991) have shown it to be expressed at 9.5d in clusters of cells arising from foregut epithelium and in cords of cells beginning to invade the septum transversum. In the experiments with α-fetoprotein, the liver was labeled at all subsequent stages (day 11 onwards), and, upon histological examination appeared to occur primarily in the endothelial cells. Hematopoietic cells appeared refractile but did not contain the hybridization grains that were visible over the a-fetoprotein positive cells.

ELF mRNA distribution in mesodermal tissues versus Alpha fetoprotein mRNA in endodermal tissue:

Since Northern analysis revealed elf expression to occur in day 11.5 heart and liver tissue, we investigated whether elf expression was restricted specifically to mesodermal tissue from the heart and the liver and compared this to the endothelial expression of α-fetoprotein. Three main regions of mesoderm can be discriminated in the developing embryo: dorsal (somitic), intermediate, and lateral. Lateral plate mesoderm comprises somatic (pleura, pericardium, peritoneum and limb bud), and splanchnic (heart-epicardium, myocardium, connective tissue and smooth muscles of viscera and blood vessels, hemangioblastic tissue, adrenal cortex and spleen). Regarding the developing heart, at day 9 (13-20 somites), this is seen to beat regularly and strongly. At this stage, the heart appears to be the only region within the embryo where the endothelial elements of the circulation are surrounded by a vessel wall. The walls of the common ventricular chamber as well as the common atrial chamber show an increasing degree of trabeculation. Of note, the space between the endothelial and myocardial elements is filled with loose mesenchyme called cardiac jelly. In situ hybridization of days 9 and 10 embryonic heart tissue using elf antisense riboprobes demonstrated high levels of labeling to both the atrial and ventricular regions. Hepatic mesenchyme also originates from lateral plate mesoderm. The septum transversum part of the hepatic mesenchyme originates from the splanchnic mesoderm of the precardiac area and this is thought to be responsible for the subsequent differentiation of hepatocytes. However, tissue explant experiments have shown that all derivatives of the lateral plate can replace hepatic mesenchyme for these later events. While these initial experiments have demonstrated migrating endoderm must interact with mesenchyme for the former to differentiate into hepatocytes (Le Douarin, 1975, supra; Houssaint, Cell Differ. 9:269-279, 1980), more recent studies investigating albumin mRNA expression as an indicator of hepatocyte differentiation, have confirmed these features: initial expression of albumin mRNA occurs during the invasion of the septum transversum, when the hepatic precursor cells clearly contact cardiac mesenchymal tissue. Similarly, primer extension analysis of albumin transcription has revealed the start site of transcription to occur at day 10.5 with a 15-20 fold increase in albumin mRNA upon liver organ formation by day 12.5. In our experiments using α-fetoprotein as a marker for differentiated hepatocytes, it was clear under high magnification, that while a-fetoprotein expression is restricted to the later endodermal component of liver development, elf expression seems to occur in the loosely organized, lighter staining mesenchymal cells--initially cardiac mesenchyme (at day 9.5), then in both cardiac and hepatic tissue (at day 10.5) and then restricted to liver tissue (day 11.5 onwards; elf expression then decreasing upon liver formation. Examination of the later histological sections (days 11 onwards) showed a diffuse distribution of grains. The resolution that was attained did not allow one to draw a firm conclusion about the identity of the hybridizing cells, although it seemed that the hybridization signal with elf was localized in the perisinusoidal cells, but not in the hepatocytes.

Distribution of elf RNA in Adult tissues, conservation in evolution:

Further characterization of elf has involved RNA analysis of adult mouse tissues. Elf hybridizes to adult liver, kidney and testis as a 2.1 Kb transcript in liver and kidney and a 2.6 Kb transcript in adult testis, in very low abundance. Genomic analysis of elf DNA from human, monkey, rat, mouse, dog, cow, rabbit, chicken and yeast indicates that elf is conserved across the species, being represented in all except rabbit DNA.

In vitro transcription and translation of elf, the latter using nuclease-treated rabbit reticulocyte lysate (promega), has revealed a 34 Kd protein, which is as predicted by the elf insert size and indicating that this insert is in frame for the coding sequence for a specific protein.

Embryonic liver explants cultures:

One of the goals of the investigations in conjunction with the present invention was to establish a functional assay for determining the developmental roles of elf and ss3 in liver formation. Mouse embryonic liver explants were cultured in our laboratory, in order to overcome the dissection and analysis of extremely small tissue sections at day 10-10.5 when the liver bud is 0.2 mm. When cultured in the complete absence of mesodermal derivatives, hepatic endoderm deteriorates rapidly. Only 2 out of 15 such liver explants survived. Hematoxylin and eosin staining showed a necrotic endoderm with no apparent signs of hepatic differentiation. When associated with the surrounding mesoderm particularly cardiac mesoderm (en bloc dissections), the endodermal cells had proliferated and invaded the mesoderm strands. Hepatocytes were seen to be organized in cords separated by sinusoids with pseudo-lobule formation. All 15 out of 15 cultures from en bloc dissections were completely viable. These studies confirm prior explant studies demonstrating the necessity of surrounding mesoderm for liver formation. Semi-quantitative RT-PCR analyses of elf, other clones ss3, 145, HNF 3β with GAPDH and α-fetoprotein as controls demonstrate increased expression during mesodermal--endodermal interactions.

Early experiments in chick embryos (Le Douarin, 1975, supra) have demonstrated that at the primitive streak stage, the prospective hepatic area is localized in the middle and in the lateral areas anterior to Hensen's node. At the head process stage, prospective liver areas coincide with cardiac areas, being concentrated in bilateral areas extending from the tip of the head process to an area slightly behind the primitive pit. Potential liver areas were tested by transplantation of pieces of tissue on the chorioallantoic membrane; liver differentiation in such explants was dependent upon the presence of cardiac tissue: no liver tissue was found without cardiac cells in the vicinity, whereas some grafts contained heart tissue without liver. After gastrulation is completed, it is during the somitic stage that the liver and heart segregate partially--the presumptive cardiac mesenchyme migrates anteriorly and venally into the cardiac fold, the prospective myocardial cells becoming incorporated in the heart anlage. Another series of experiments using carbon particle labeling, radiodestruction and coelomic transplantation of pieces of blastoderm showed liver endodermal and mesodermal areas which am superimposed during the early embryonic stages evolve differently later on.

Tissue explant studies have revealed that in normal liver development, hepatocyte differentiation and the formation of liver lobes is entirely dependent upon the mesodermal component which then becomes progressively colonized by the growing endoderm hepatic cords (see Le Douarin, 1975, supra). These stimulating properties of the cardiac, and then, hepatic mesenchyme have been demonstrated to begin at the 5 somite stage and last throughout embryonic life. The findings set forth herein show that elf is expressed in early cardiac mesoderm, with subsequent expression being limited to hepatic mesoderm, revealing this to be a novel marker for the mesodermal component of liver development. Of note, in normal development, pure liver mesenchyme is never observed. That these explant studies have demonstrated expression of elf, indicates that the elf protein will be useful in identifying and studying such interactions between mesoderm and foregut endoderm.

    ______________________________________     Summary of events during hepatocyte formation indicating     a role for elf     Embryonic     ______________________________________     Stage   endodermal cell hypertrophy                                    |             |             | elf      day 9.5             change in cell polarity                                    |expression             |     day 10.5             invasion and migration into surrounding             mesenchyme             |     day 11.5             pseudolobule formation, cords of             hepatocytes, early sinusoidal formation             |     day 14.5             hematopoietic foci and fully differentiated             fetal hepatocytes     ______________________________________

Sequence analysis has shown elf to bear 80% identity to β-fodrin, a non erythroid β-spectrin. β-spectrins have been implicated in numerous functions including the maintenance of cell surface polarity of cells (Nelson et al., J. Cell. Biol. 108:893-902, 1989); the maintenance of cell-cell junctions (Thomas et al., Development 120:2039-2050, 1994, Luna et al., Science 258:955-964, 1992); β-spectrins contain binding sites for other proteins, such as ankyrin and actin (Hu et al., J. Biol. Chem. 267:18715-18722, 1992; Speicher et al., Nature 311:177-180, 1984). Smaller isoforms β-spectrins have been well described. For instance, a 4.0 Kb muscle tissue transcript is thought to encode a previously reported β-spectrin from clustered acetylcholine receptors. Similarly for elf, the missing domains may be replaced through alternate exon usage to generate proteins with unique functions. A function for elf thus appears to be in the assembly and maintenance of specific domains on the cell surface--towards establishing hepatocyte polarity and thus differentiation.

Spectrins have also been shown to be conserved throughout evolution and am developmentally regulated. These results demonstrate that in keeping with brain β-spectrin (β-G spectrin), elf is also expressed in a tissue and stage specific manner and is conserved throughout evolution (Hu et al., J. Biol. Chem. 267:18715-18722, 1992; Zimmer et al., Brain Res. 594:75-88, 1992; Leto et al, Mol. Cell Biol. 8:1-9, 1988). Elf expression occurs in a gradient-like manner and close examination of Brain β-G spectrin has demonstrated similar gradient patterns, suggesting that a sudden on-off phenomenon at specific time points is simplistic. That elf is maximally expressed at day 10-11 suggests that it has an important function at this time, which continues, although to a lesser extent, with the later stages. For instance, it is conceivable that elf by conferring cell polarity mark the first overt sign of hepatocyte differentiation. Therefore, like Drosophila β-H spectrin, elf may play a role in facilitating a "velcro-like" joining of neighboring cell membranes as they extend (Thomas et al., Development 120:2039-2050, 1994). In this way elf may mark the polarization of the surrounding mesodermal cells, enabling foregut endodermal cells to invade this area and differentiate into hepatocytes. Molecular markers have been invaluable in the dissection of inductive events in embryological studies (New et al., Curr. Opin. Genet. Dev. 1:196-203, 1991; Sive, Genes Dev. 7:1-12, 1993). For instance, in Xenopus, Epi 1, an antibody specific for epidermis, has been used to elucidate the role of the blastopore lip in the neural induction process (Savage et al., Dev. Biol. 133:157-168, 1989). Similarly activins (regulating keratin) (Asashima et al., P.N.A.S. 88:6511-6514, 1991), vg-1 (Thomsen et al., Cell 63:485-493, 1990) and other genes belonging to the TGF-β family, as well as wnt and bFGF families represent components of the cascade leading to the commitment to particular mesodermal fate. For instance, vg-1, originally isolated by differential screening is to cells inducing embryonic mesoderm, the posttranslational processing of Vg-I precursor protein on the future side of the embryo being a key step in generating dorsal mesoderm and body axis in Xenopus (Thomsen et al., Cell 74:433-441, 1993). Similarly, in isolating putative inductive agents required for liver formation, a key step is the identification of mRNAs localized to cardiac/liver mesenchyme: elf and its regulatory genes will help to elucidate this area.

More recently, cell-cell interactions have been shown to be important for several cell fate decisions. In C. elegans for instance, lin-12 and glp-1 have been shown to encode transmembrane proteins mediating intracellular communication, and are required for the specification of several anterior fates. In Drosophila, the establishment of secondary epithelia which are the result of a mesenchymal-epithelial transition, is thought to be dependent upon two separate adhesions systems: direct interactions between the developing midgut endoderm and the visceral mesoderm on one hand and, adhesive interactions between the epithelial cells themselves on the other. While the latter cell-cell interaction is thought to be controlled by shotgun, control of apicobasal polarity is thought to be caused by genes such as crumbs and stardust (Tepass et al, Cell 61:787-799, 1990). Although it is known that the biogenesis of cell surface polarity in hepatocyte formation is an early event, implying that the mechanisms for sorting plasma membrane molecules are functional at an early point, genes involved in cell signaling leading to cell fate in liver development have not been defined to date. The identification of such genes would give tremendous insight into the cell-cell interactions involved in foregut endodermal cell migration and subsequent morphogenesis of the liver as an organ. These studies establish the principle that specific mesoderm mRNAs are localized in a way that guarantees their subsequent segregation to specific mesodermal tissue, in this case the presumed mesodermal component of the liver as demonstrated by embryonic explant studies (Le Douarin, 1975).

Cloning and sequencing of elf:

All embryonic liver was obtained from matings of random-bred ICR mice (Harlan). The plug date was designated as Day 0 and embryos collected at days 10.0, 11.5 and 12.5 p.c.; these were staged by morphological criteria (Theiler, The House Mouse, New York: Springer-Verlag, 1989). The livers were dissected, pooled and lysed. To prepare cDNA libraries, RNA was isolated (Chomczynski et al., Analyt. Biochem. 162:156-159, 1987) and poly(A)+ RNA selected using oligo(dT)-cellulose (Collaborative Research Type 3). 1 to 5 mg of poly(A)+RNA were used in the preparation of oligo(dT)- primed cDNA libraries. cDNA library construction of days 11.5 and 12.5 embryonic liver was carried out by conventional techniques (Gubler et al., Gene 25:263-269, 1983), and the day 10.0 and adult mouse liver using the Stratagene Unizap cDNA library kit. Two subtracted libraries were then constructed (Schweinfest et al., Genet. Anal. Tech. Appl. 7:64-70, 1990). The resulting subtracted libraries comprised 64 clones (11.5-12.5), and 110 clones (10.5-11.5). The process involved: (a) Biotinylation: fifty micrograms of cDNA from day 12.5 liver library at 10mg/ml were biotinylated in HE buffer (10 mM Hepes, pH 7.5, 1 mM EDTA, Clontech Labs.); (b) Subtraction was then done by the streptavidin-phenol extraction: the streptavidin-biotin hybrid duplexes represent common gene products which selectively partition into the phenol interface, leaving the unique, subtracted single stranded cDNA in the aqueous phase. After synthesis of second strand DNA and overnight precipitation, one tenth of the DNA was used to transform competent XL Blue cells. Transformation using all the subtracted DNA led to the identification of 174 recombinant colonies. Purification of bacteriophages, preparation of DNA were carried out by the stratagene in vivo excision protocol. Plasmid DNA was sequenced using 77 DNA polymerase (Sanger et al., J. Mol. Biol. 143:161-178, 1980).

Sequence analysis:

The NCBI non-redundant (nr) and EST databases were searched using the blastp2 and blastn2 programs, which permit gapped alignments (Altschul et al., Methods in Enzymology 256:460-480, 1996), with the default parameters and elf protein or nucleotide sequences as queries.

RNA preparation and analysis:

Embryos were collected at day 10.0, 11.5 and 12.5 p.c. Embryonic livers were dissected in Dulbecco's modified Eagle's medium (high glucose) and 20 mM Hepes pH 7.3. The livers for the specific stages were pooled and total RNA isolated (Chomczynski et al., supra). 10 micrograms of RNA were electrophoresed on a 1% formaldehyde gel and transferred onto Hi-bond nylon membrane (Amersham) using standard procedures (Sambrook et al., 1989, supra). Radioactive, ³² P-labeled probes were synthesized by random primer methods (Feinberg et al., Analyt. Biochem. 137:266-267, 1984) and hybridized to the Nylon filters. Filters were washed at high stringency with a final wash in 0.2×SSC (30 mM NaCl, 3 mM sodium citrate, pH 7.4) 0.5% Sodium Dodecyl Sulfate at 65° C. for 60 minutes. Filters for each probe were stripped and rehybridized with other probes to confirm that no cross hybridization signals were obtained under initial screening conditions. These filters were then autoradiographed with intensifying screens at -70° C.

In Situ Analysis:

In situ analysis was performed for elf (Cox et al., Dev. Bio. 100:197-206, 1989). The RNA probes were synthesized and labeled with ³⁵ S-UTP (400 Ci/mmole) via the T7 or SP6 promoter for RNA polymerase. Sense or antisense probes were added to the appropriate sections, mounted, sealed with rubber cement and incubated at 50° C. overnight. After incubation, sections were washed with 50% formamide/5×SSC/10 mM DTT (50° C.; 2×30 min.) followed by 4×SSC/TE, incubated with RNase A (20 mg/ml) and RNase TI (500 U/ml; 37° C. 30 min), rinsed again with 4×SSC/TE (37° C., 30 min), twice 2×SSC (25° C., 15 min), twice in 0.1×SSC (25° C., 15 min), dehydrated with an ethanol series (containing 0.3 M ammonium acetate) and air dried. For autoradiography, slides were dipped in NTB 2 emulsion diluted 1:1 with 2% glycerol in water and dried. Exposure times were from @ weeks to four months. The emulsion was developed according to manufacturer's directions.

In Vitro Translation of elf:

Bluescript containing elf was transcribed with T7 RNA polymerase using the in Vitro Eukaryotic Translation kit and MCAP mRNA Capping kit (Stratagene). The RNA transcript was translated in vitro into protein for 90 minutes in the presence of ³⁵ S!methionine using nuclease-treated rabbit reticulocyte lysate (Promega) and run on 4% denaturing polyacrylamide gels.

Liver explant cultures:

Mouse embryos were obtained from Harlan ICR mice. The age of the embryos was determined by days post appearance of the vaginal plug (day 0). The embryos were further characterized by the number of somites. Isolation of mouse hepatic endoderm, liver buds and mesoderm (en bloc dissection) was as follows: during the 10th day of gestation, the liver bud becomes evident as a thickening of the ventral wall of the foregut, near the origin of the yolk stalk. This ventral endoderm was then either taken alone and cultured, or alternatively with the surrounding mesoderm: the portion of the embryo between the otocyst and the umbilical region. Organ culture: Embryos were placed into nucleopore filters in a humid chamber as described (Houssaint, 1980, supra) and cultured for 48 hours or 96 hours. Microscopy: The explants were fixed as in the in situ hybridization protocols, and RNA isolated as described above. 7 mm sections were stained with hematoxylin, eosin and periodic acid schiff (PAS) for glycogen, an indicator of differentiated hepatocytes. For RNA analysis, semiquantitative RT-PCR was performed.

    __________________________________________________________________________     #             SEQUENCE LISTING     - <160> NUMBER OF SEQ ID NOS: 18     - <210> SEQ ID NO 1     <211> LENGTH: 5434     <212> TYPE: DNA     <213> ORGANISM: Mus musculus     <220> FEATURE:     <223> OTHER INFORMATION: For all n's in this - # sequence, n=(a or g or           or t)     <220> FEATURE:     <221> NAME/KEY: CDS     <222> LOCATION: (1674)..(2069)     - <400> SEQUENCE: 1     - tcgggaaang attgatttgg ccncctcggn aaggcntttt attttgcnnc aa - #ggagggcc       60     - cggggggttt ccaaccnaaa taaaattttt tttcggatcc cgggggtttc ct - #cagggagt      120     - tggggaattt tactttgaaa gcagatnttt cngagntccg ggtagctntc ca - #ataactnt      180     - ttgtcatcat tgccagacgg cagatcaagg atgccttcgg tttacccgtg ct - #gttcagag      240     - aacggctttt ggaagattga ttttaagtta tttaacagtc acagacaggt gt - #catntntg      300     - gagaatagag gcaagtccgc ggtgagggat gaagcaggag agattagggg aa - #ggcagaca      360     - ggactgctgg gccaaggaag ctgtgctgat ttgagcacag tgggaattca cg - #tacgcaat      420     - ttcaaaggct ttagtggtaa attctgaagc tcagatgcag gcaagaccca ag - #aggatagt      480     - gtacacagag agaagagggt cntcaggatc gtgcgtagag tggagagagc cc - #caaaggca      540     - ggagggaaga gcctcagtga ttacttaggg atgagggaga gaagaaaaaa gg - #ttcttgca      600     - aggtgtgggg tcttccaaat tcaggagttc actgccatat agagaaggtg ta - #gcgggtga      660     - aaggggccat gtgatgagga tggcaagcaa ggctgtggcg cagatgacga ga - #tgcctggg      720     - tcgggaggtc aggggagacc caggattggg gtcacctgtg tctgcgcaga gg - #ggaagcca      780     - ccctgcaact ggcccagcac tgagtccaga ggaaaatgag gcagaggaca aa - #ccagagct      840     - tcggagacta agtgcaggta gggcgcgggc ggagcgtgag gagggcagcg ga - #ccacgcga      900     - gaggcctcga aggccaccgg acccgcgtcc gagagtctga gggccctgcc ca - #cacctgcg      960     - tggccccctc cccagaggcc acactccaag gccaccctag aacccgtctg tc - #tgctcaag     1020     - cccttgcaaa agacgtctgc gcagaggggg cgtggcaggc gtgctgtcac tc - #acggcctg     1080     - ttagccaatc cacgagtgcg cccctccccg gagagggtgc gcggagggcc cg - #cccccgcc     1140     - gccaccgcgg gtgtgaggag gccaggctgg cgcggctccc tccgcccggc ag - #ccttgcca     1200     - ggtaaccggg ttcggcggga gggctggggg tcgcgcagcc ccctcgctcc ct - #gggaggcg     1260     - tgcacactgc cgcggcgggt cccgtgtggg ccggaggccc gtgcgcgcgt cg - #gaccgacg     1320     - ggccgcagcc tgtgggcggg gttgcgtgcg tgacgggcgg ccgtgccccg cg - #ttgtgtca     1380     - ggcctgcgcg gggaaagctc ggccgaaccg aggtgtccag gtccgcccgc tg - #cggcctgc     1440     - cccgggttgc ggggcgcagg cgcggcggtg ggcgggggtc gtccccagga gc - #gtctttgt     1500     - tcccggcgcg ctgagggcgg agcctcaccc cgccccgccc ccgcgctcag tc - #cccgcccc     1560     - gcgtccgccc gcaggagctg ccaccgggtc ccgctggcct ccccggccgc cg - #ccaccgcc     1620     - tccgcctccg ccgctccggg cccgccggct tgcgtcgccg aggtcgctgc ag - #c atg     1676     #     Met     #       1     - gcg ggc gtc gcg acc ccc tgc gcc aac ggc tg - #c ggg cct ggc gca ccc     1724     Ala Gly Val Ala Thr Pro Cys Ala Asn Gly Cy - #s Gly Pro Gly Ala Pro     #              15     - tcc gaa gcc gag gtg ctg cac ctc tgc cgc ag - #c ctc gag gtg ggc acc     1772     Ser Glu Ala Glu Val Leu His Leu Cys Arg Se - #r Leu Glu Val Gly Thr     #         30     - gtc atg act ttg ttc tac tcc aag aag tcg ca - #g cgg cca gaa cgg aag     1820     Val Met Thr Leu Phe Tyr Ser Lys Lys Ser Gl - #n Arg Pro Glu Arg Lys     #     45     - acc ttc cag gtc aag ttg gag acg cgc cag at - #c aca tgg agc cgc ggc     1868     Thr Phe Gln Val Lys Leu Glu Thr Arg Gln Il - #e Thr Trp Ser Arg Gly     # 65     - gcg gac aaa atc gag ggg tcc agt aag tgc gc - #c cca ctc cgg cct gcc     1916     Ala Asp Lys Ile Glu Gly Ser Ser Lys Cys Al - #a Pro Leu Arg Pro Ala     #                 80     - tcg cgc ctg ccc gcc tcc caa aca ctt ggg ca - #a act ttc ggg cct cgc     1964     Ser Arg Leu Pro Ala Ser Gln Thr Leu Gly Gl - #n Thr Phe Gly Pro Arg     #             95     - gcc tgg cgc ccc gtc tcc gcc cag tcc ctg gt - #g gtc act ctg ggg cgg     2012     Ala Trp Arg Pro Val Ser Ala Gln Ser Leu Va - #l Val Thr Leu Gly Arg     #       110     - gtg gag ggg ggc atc cgg gtc ttg gat cac ct - #g ata gga cac ccc ctc     2060     Val Glu Gly Gly Ile Arg Val Leu Asp His Le - #u Ile Gly His Pro Leu     #   125     - ccc cag tag ggggggagtg ttccaggcac tttgccctga ggcctaaga - #g     2109     Pro Gln     130     - tcctcactgg ttggacaagt ggagtgggat tccggccctt agcatcgggc gg - #ctgtcagt     2169     - ggctgtgagg ggaagccaag acagggaccc cctcatccaa cctgagaacc tg - #gggaaccg     2229     - acaagatctt cctgcccact gccatttctc cagagtgtgc tgtctgtgaa aa - #ctcctaag     2289     - agctccggga tgggcttatt ggcgcaagaa cctttggaat cctcatgtag aa - #cttaggca     2349     - gatgttgggg tagggctggt tgtgaagcag agccctactc atctcccctc tt - #ctttggga     2409     - ggatggggta tgaaagctaa aaccgtgact gcttccccct cccatgtccc gt - #ggatgggt     2469     - tttttttttt tttttttttg ccccagatct gaattttgga ggtccatggt gc - #taggcagc     2529     - catccaaagc tagagccatg gctcctttgc ccttgcagca tataacaagg ag - #cttgcatt     2589     - cagaaaggtt ccctggcctt gggttttggg gtccagccct ttgtgttgga tg - #ttctcgtg     2649     - accacagggt agcccagagt tgctcctctg gtttcctgtc gtacccttcc ca - #aacctgag     2709     - tgtggtgggt ttacacacaa gtctctggtg ggagaagtaa gtcaggagtt tt - #gagaaacc     2769     - tcggctcttt ctgatagtca ttttcctcgg tgtgaggcag gatgaggagt ct - #ttgcaact     2829     - ccaggctttg agatgtttct tacaagaacc cccaaagagt ctatggttga ag - #ggacctag     2889     - cctaagagcc aggtctgtgt tagagaaggg ggggtggtgt caggaagtaa ca - #acggagag     2949     - aaggtcccac agatcttcct ggggatggtg tacatgtgtg tcgatgggtg ag - #gagatgag     3009     - gaggaaggaa ggtttctgtg gtaagacagc catcctcaac tacaaacttc ag - #gtctgaca     3069     - gaattggccc ttaaccatca ccagtgccca tcagccctgg cctccgctgg aa - #gaacattt     3129     - cagtgatttt cagtgttggg ggatggaact gcagacagtt ccggtagtcc tg - #agacatca     3189     - ctcagacatc aggttgcagg catggcattt tacgtttgta gtatttcctg tg - #tttaagtg     3249     - gtggcattag ttccccggta gctagctctt ggtaacagct gcactgtaaa cc - #gtgtgtgt     3309     - agcccagtag tggaagatag ctatggtatt tgaagccagt gtgttagctg ta - #cgtcaccc     3369     - agccaggtgc tttccctctc ggagcctcgg ttcctctgta agttagcaga ag - #tatattta     3429     - ctataaatgg tcacttttgg aagtgagata gttggtgtaa agtaagcaaa ct - #aaatatgt     3489     - aatagatgcg agcagagacg ttacagaagt ttaagaacca gttattagta gc - #agtagcta     3549     - tggtagatgc ttgtcctcct agaccctggg atggggcttc tgagggaggt ct - #aatgtggc     3609     - tgttagaaaa agaaagggct ctgagggagg agggccgaga gagggtcccg tt - #ctccttaa     3669     - ttgcattacc caggataaaa gaggaaactc ttgttttgcc gtacatcgtt ta - #cccttctg     3729     - ttcacctgtc atgtaagatg agtttctatg tttggaattt tgtacattgg at - #gccattgt     3789     - gagttggggc ctggacagaa agaagggact tagagacaga accatccagt cc - #gttttgtc     3849     - tcacttgggt ctttgaggat gggtggcagg aatacagagg acgtcacctt tc - #cagaccca     3909     - caaaagtcac ccagagatat gcatgttttc attgggcccg accctgtgat tt - #ttggggtc     3969     - cagaatgaag gctgcagact agcctgtgtg gacttcatac cttgtaaatg ga - #gcccacca     4029     - ccgaagccct gccccacttc tgctggaatg cacctcactg cctttgtggg tt - #cccaaacc     4089     - tgcagcctcc tgcagattgt gaaaaggatt gagttgccag ctgggtccct ac - #tgtctggt     4149     - ctcttgttca gatgcctcag gtatttgact ttttgctgat aaccttatcc ct - #acctgaag     4209     - ccaggccaga gagaaagact gccgctgtct gccctcaggg tgctcacgga ac - #acaacgac     4269     - aggctgactg ccatttccta aatcttgagt tctctcactg tgacacctgt ga - #aactagtt     4329     - agcaccttct gatgtctaag gcagcggtct acttgagaag tgctttggtg ct - #gtttggtt     4389     - gtgtgactga agtcaggctg gtgtctggca tttatgttgc agaatttagt ga - #gttaaaag     4449     - cagccataga cttcctgccc agtgctaaac agacttttca ctctgctgca gg - #ctagtcct     4509     - cagaggactc tgctcccagg ttgtgttggt ggtaggcctt ggtctcctgt tt - #tctgtagc     4569     - ctttgttgcc ccttgtgaag agaaacctcc atgtttaggt ggtatttaca gg - #cagagacc     4629     - tccatcttca tcaaagacgc cttcctaggc tttccatatg taatgcctgt ag - #tgagatgg     4689     - ctcagaccta ttcttcgtga ggttgtccag ttaaggacca ctgttggcat ag - #tagctcca     4749     - gtagagactc taaagctatg ttgttattgt ggtgaggatt gcagtaccaa gg - #ggctggct     4809     - ctgagagtag gtccgtggca cctaagaatt gtctgcacat gtccctcaag ga - #ttcctttt     4869     - ngctggccca cagtgagaga gcagcagaaa gcatgcgcct ggatctaaga aa - #ggttaatg     4929     - aaaccatggt acctatggga gctttacaac ctgggcttct gtctccggta gc - #catttcta     4989     - aaaganatta tgaaattgtg gtagattgaa agatgttcct tactattcct tt - #acatcctg     5049     - aggatcacga aagatttgct ttcagtattc ctactattaa ttttaaagaa cc - #tatgaaaa     5109     - gatatcaatg gacagttctt ccacaaggca tggctaataa tcctacctta tg - #tcaaantt     5169     - gtggcacaac cattcacctg tgagacacaa tgactatgac tactcntcnt ga - #tgatgatg     5229     - angatgatga gatgatgatg atgatgatga tgacacacan gatagagatg at - #tctaangc     5289     - ggaaanatcc cgactgcttt ncttaaaatt accnncctnc gaaaagatta aa - #cccgaaag     5349     - gtcaccgatc tatatttngt ttaantnata ccgtttccca aaattttncg ga - #cctnaant     5409     #             5434 atgn tcccc     - <210> SEQ ID NO 2     <211> LENGTH: 131     <212> TYPE: PRT     <213> ORGANISM: Mus musculus     - <400> SEQUENCE: 2     - Met Ala Gly Val Ala Thr Pro Cys Ala Asn Gl - #y Cys Gly Pro Gly Ala     #                 15     - Pro Ser Glu Ala Glu Val Leu His Leu Cys Ar - #g Ser Leu Glu Val Gly     #             30     - Thr Val Met Thr Leu Phe Tyr Ser Lys Lys Se - #r Gln Arg Pro Glu Arg     #         45     - Lys Thr Phe Gln Val Lys Leu Glu Thr Arg Gl - #n Ile Thr Trp Ser Arg     #     60     - Gly Ala Asp Lys Ile Glu Gly Ser Ser Lys Cy - #s Ala Pro Leu Arg Pro     # 80     - Ala Ser Arg Leu Pro Ala Ser Gln Thr Leu Gl - #y Gln Thr Phe Gly Pro     #                 95     - Arg Ala Trp Arg Pro Val Ser Ala Gln Ser Le - #u Val Val Thr Leu Gly     #           110     - Arg Val Glu Gly Gly Ile Arg Val Leu Asp Hi - #s Leu Ile Gly His Pro     #       125     - Leu Pro Gln         130     - <210> SEQ ID NO 3     <211> LENGTH: 6960     <212> TYPE: DNA     <213> ORGANISM: Mus musculus     <220> FEATURE:     <221> NAME/KEY: CDS     <222> LOCATION: (333)..(6794)     - <400> SEQUENCE: 3     - cctgcgtcct tcctcctttt cctccttccc tcctccctcc cgggtaattt at - #ttctagct       60     - tccaggcaag ggccacacaa ggaaggaaat ccacagggga ttagatgccg gg - #gtggtaac      120     - tccaccaggc taggttggac tctgcagcca acttcctatc agatcaccct gc - #acctattt      180     - ccgacccgac cggaatgcga ctggcttgag gtccagccct ttcgcctggg cg - #ggagcaga      240     - gccgcggaag ctgcttggag ttggatgggg gtaggaaggg gctggagcgg ga - #atcctacg      300     - atgcaactgg cctgggccta aggttgggca ta atg gag ttg cag - # agg aca tcc      353     #Met Glu Leu Gln Arg Thr Ser     #  1               5     - agc gtt tca ggg ccg ctg tcg ccg gcc tac ac - #c ggg cag gtg cct tac      401     Ser Val Ser Gly Pro Leu Ser Pro Ala Tyr Th - #r Gly Gln Val Pro Tyr     #         20     - aac tac aac caa ctg gag gga aga ttc aaa ca - #g ctc caa gat gag cgt      449     Asn Tyr Asn Gln Leu Glu Gly Arg Phe Lys Gl - #n Leu Gln Asp Glu Arg     #     35     - gaa gct gta cag aag aag acc ttc acc aag tg - #g gtc aat tcc cac ctt      497     Glu Ala Val Gln Lys Lys Thr Phe Thr Lys Tr - #p Val Asn Ser His Leu     # 55     - gca aga gtg tcc tgc cga atc aca gac ctg ta - #c acg gac ctt cga gat      545     Ala Arg Val Ser Cys Arg Ile Thr Asp Leu Ty - #r Thr Asp Leu Arg Asp     #                 70     - gga cgg atg ctc atc aag cta ctg gag gtc ct - #c tct gga gag agg ctg      593     Gly Arg Met Leu Ile Lys Leu Leu Glu Val Le - #u Ser Gly Glu Arg Leu     #             85     - cct aaa ccc act aag gga cgg atg cgg atc ca - #c tgt ctg gag aat gtc      641     Pro Lys Pro Thr Lys Gly Arg Met Arg Ile Hi - #s Cys Leu Glu Asn Val     #        100     - gac aag gct ctt caa ttc ctg aaa gag cag ag - #a gtc cat ctt gag aac      689     Asp Lys Ala Leu Gln Phe Leu Lys Glu Gln Ar - #g Val His Leu Glu Asn     #   115     - atg ggc tcc cat gac att gtg gat gga aac ca - #c cgg ctg acc ctc ggc      737     Met Gly Ser His Asp Ile Val Asp Gly Asn Hi - #s Arg Leu Thr Leu Gly     120                 1 - #25                 1 - #30                 1 -     #35     - ctc atc tgg aca att att ctg cgc ttc cag at - #c cag gat att agt gtg      785     Leu Ile Trp Thr Ile Ile Leu Arg Phe Gln Il - #e Gln Asp Ile Ser Val     #               150     - gag act gaa gat aac aaa gag aaa aag tct gc - #t aag gat gca ttg ctg      833     Glu Thr Glu Asp Asn Lys Glu Lys Lys Ser Al - #a Lys Asp Ala Leu Leu     #           165     - ctg tgg tgc cag atg aag aca gct ggg tac cc - #c aat gtc aac att cac      881     Leu Trp Cys Gln Met Lys Thr Ala Gly Tyr Pr - #o Asn Val Asn Ile His     #       180     - aat ttc acc act agc tgg agg gat ggc atg gc - #c ttc aat gca ctg ata      929     Asn Phe Thr Thr Ser Trp Arg Asp Gly Met Al - #a Phe Asn Ala Leu Ile     #   195     - cat aaa cat cgg cct gac ctg ata gat ttt ga - #t aaa ctg aag aaa tct      977     His Lys His Arg Pro Asp Leu Ile Asp Phe As - #p Lys Leu Lys Lys Ser     200                 2 - #05                 2 - #10                 2 -     #15     - aat gca cac tac aat ctg cag aat gca ttt aa - #c ctg gca gag cag cac     1025     Asn Ala His Tyr Asn Leu Gln Asn Ala Phe As - #n Leu Ala Glu Gln His     #               230     - ctt ggc ctc act aaa ctg tta gac cct gaa ga - #t atc agt gtg gac cac     1073     Leu Gly Leu Thr Lys Leu Leu Asp Pro Glu As - #p Ile Ser Val Asp His     #           245     - cct gat gag aag tct atc atc aca tac gtg gt - #g act tac tac cac tac     1121     Pro Asp Glu Lys Ser Ile Ile Thr Tyr Val Va - #l Thr Tyr Tyr His Tyr     #       260     - ttc tcc aag atg aag gcc ttg gct gtc gaa gg - #a aag cgc att gga aag     1169     Phe Ser Lys Met Lys Ala Leu Ala Val Glu Gl - #y Lys Arg Ile Gly Lys     #   275     - gtg ctt gat aat gct ata gaa aca gag aaa at - #g att gag aag tac gag     1217     Val Leu Asp Asn Ala Ile Glu Thr Glu Lys Me - #t Ile Glu Lys Tyr Glu     280                 2 - #85                 2 - #90                 2 -     #95     - aca ctt gct tct gac ctt ctg gag tgg att ga - #a caa acc atc atc atc     1265     Thr Leu Ala Ser Asp Leu Leu Glu Trp Ile Gl - #u Gln Thr Ile Ile Ile     #               310     - cta aac aac cgc aaa ttt gct aat tca ctg gt - #t ggg gtc caa cag cag     1313     Leu Asn Asn Arg Lys Phe Ala Asn Ser Leu Va - #l Gly Val Gln Gln Gln     #           325     - ctc caa gca ttc aac acg tac cgc aca gtg ga - #g aaa cca cct aag ttt     1361     Leu Gln Ala Phe Asn Thr Tyr Arg Thr Val Gl - #u Lys Pro Pro Lys Phe     #       340     - act gag aag ggg aat ttg gag gtg ctc ctt tt - #c gcg att cag agc aag     1409     Thr Glu Lys Gly Asn Leu Glu Val Leu Leu Ph - #e Ala Ile Gln Ser Lys     #   355     - atg cga gcg aat aat cag aag gtc tac atg cc - #c cgc gag ggg aag ctc     1457     Met Arg Ala Asn Asn Gln Lys Val Tyr Met Pr - #o Arg Glu Gly Lys Leu     360                 3 - #65                 3 - #70                 3 -     #75     - atc tct gac atc aac aag gcc tgg gaa aga ct - #g gaa aaa gca gaa cat     1505     Ile Ser Asp Ile Asn Lys Ala Trp Glu Arg Le - #u Glu Lys Ala Glu His     #               390     - gag aga gaa ctg gct ctg cgg aat gag ctc at - #a cgg cag gaa aaa ctg     1553     Glu Arg Glu Leu Ala Leu Arg Asn Glu Leu Il - #e Arg Gln Glu Lys Leu     #           405     - gaa caa ctc gcc cga aga ttt gat cgc aag gc - #a gct atg agg gag aca     1601     Glu Gln Leu Ala Arg Arg Phe Asp Arg Lys Al - #a Ala Met Arg Glu Thr     #       420     - tgg ctg agt gaa aac cag cgt ctt gtg tct ca - #g gac aac ttt gga ttt     1649     Trp Leu Ser Glu Asn Gln Arg Leu Val Ser Gl - #n Asp Asn Phe Gly Phe     #   435     - gac ctt ccc gct gtt gag gct gct acc aaa aa - #a cac gag gcc att gag     1697     Asp Leu Pro Ala Val Glu Ala Ala Thr Lys Ly - #s His Glu Ala Ile Glu     440                 4 - #45                 4 - #50                 4 -     #55     - aca gac atc gct gca tat gaa gaa cga gtt ca - #g gcc gtg gtg gct gtg     1745     Thr Asp Ile Ala Ala Tyr Glu Glu Arg Val Gl - #n Ala Val Val Ala Val     #               470     - gcc agg gaa ctt gaa gcc gag aac tac cat ga - #c atc aag cgc atc aca     1793     Ala Arg Glu Leu Glu Ala Glu Asn Tyr His As - #p Ile Lys Arg Ile Thr     #           485     - gcg agg aag gac aat gtc atc cgg ctc tgg ga - #a tac ttg ctg gaa ctg     1841     Ala Arg Lys Asp Asn Val Ile Arg Leu Trp Gl - #u Tyr Leu Leu Glu Leu     #       500     - ctc agg gcc agg agg cag cgt ctt gag atg aa - #c ctg gga ttg caa aag     1889     Leu Arg Ala Arg Arg Gln Arg Leu Glu Met As - #n Leu Gly Leu Gln Lys     #   515     - ata ttc cag gaa atg ctt tat att atg gac tg - #g atg gat gaa atg aag     1937     Ile Phe Gln Glu Met Leu Tyr Ile Met Asp Tr - #p Met Asp Glu Met Lys     520                 5 - #25                 5 - #30                 5 -     #35     - gtg cta ttg ctg tct caa gac tat ggc aaa ca - #c tta ctt ggt gtt gaa     1985     Val Leu Leu Leu Ser Gln Asp Tyr Gly Lys Hi - #s Leu Leu Gly Val Glu     #               550     - gac ctg tta cag aag cat gcc ctg gtt gaa gc - #a gac att gca atc caa     2033     Asp Leu Leu Gln Lys His Ala Leu Val Glu Al - #a Asp Ile Ala Ile Gln     #           565     - gca gag cgt gta aga ggt gtg aat gcc tct gc - #c cag aag ttt gca aca     2081     Ala Glu Arg Val Arg Gly Val Asn Ala Ser Al - #a Gln Lys Phe Ala Thr     #       580     - gat ggg gaa ggc tac aag cca tgt gac ccc ca - #g gta att cga gac cgt     2129     Asp Gly Glu Gly Tyr Lys Pro Cys Asp Pro Gl - #n Val Ile Arg Asp Arg     #   595     - gtt gcc cac atg gag ttc tgc tat caa gag ct - #t tgt cag ctg gct gcc     2177     Val Ala His Met Glu Phe Cys Tyr Gln Glu Le - #u Cys Gln Leu Ala Ala     600                 6 - #05                 6 - #10                 6 -     #15     - gag cgt agg gct cgc ctg gaa gag tcc cgt cg - #c ctc tgg aag ttc ttc     2225     Glu Arg Arg Ala Arg Leu Glu Glu Ser Arg Ar - #g Leu Trp Lys Phe Phe     #               630     - tgg gag atg gca gaa gag gaa ggc tgg ata cg - #a gag aag gaa aag atc     2273     Trp Glu Met Ala Glu Glu Glu Gly Trp Ile Ar - #g Glu Lys Glu Lys Ile     #           645     - ctg tcc tct gat gat tac ggg aaa gac ttg ac - #c agt gtc atg cgc ctg     2321     Leu Ser Ser Asp Asp Tyr Gly Lys Asp Leu Th - #r Ser Val Met Arg Leu     #       660     - ctg agc aag cac cgg gca ttt gag gat gag at - #g agt ggc cgt agt ggc     2369     Leu Ser Lys His Arg Ala Phe Glu Asp Glu Me - #t Ser Gly Arg Ser Gly     #   675     - cat ttt gag cag gcc att aaa gaa ggt gaa ga - #c atg att gca gag gaa     2417     His Phe Glu Gln Ala Ile Lys Glu Gly Glu As - #p Met Ile Ala Glu Glu     680                 6 - #85                 6 - #90                 6 -     #95     - cac ttt gga tcg gaa aag atc cgt gag aga at - #c att tat atc cgg gag     2465     His Phe Gly Ser Glu Lys Ile Arg Glu Arg Il - #e Ile Tyr Ile Arg Glu     #               710     - cag tgg gcc aac ctg gaa cag ctc tca gcc at - #t agg aag aag cgc cta     2513     Gln Trp Ala Asn Leu Glu Gln Leu Ser Ala Il - #e Arg Lys Lys Arg Leu     #           725     - gag gaa gcc tca tta ctg cac cag ttc cag gc - #t gat gct gat gat att     2561     Glu Glu Ala Ser Leu Leu His Gln Phe Gln Al - #a Asp Ala Asp Asp Ile     #       740     - gat gct tgg atg tta gat ata ctc aag att gt - #c tcc agc aat gat gtg     2609     Asp Ala Trp Met Leu Asp Ile Leu Lys Ile Va - #l Ser Ser Asn Asp Val     #   755     - ggc cat gat gag tac tcc acg cag tct ctg gt - #c aag aag cat aaa gat     2657     Gly His Asp Glu Tyr Ser Thr Gln Ser Leu Va - #l Lys Lys His Lys Asp     760                 7 - #65                 7 - #70                 7 -     #75     - gta gca gaa gag atc acc aac tgc agg ccc ac - #t att gac aca ctg cat     2705     Val Ala Glu Glu Ile Thr Asn Cys Arg Pro Th - #r Ile Asp Thr Leu His     #               790     - gag caa gcc agt gcc ctt cca caa gca cat gc - #a gag tct cca gat gtg     2753     Glu Gln Ala Ser Ala Leu Pro Gln Ala His Al - #a Glu Ser Pro Asp Val     #           805     - aag ggc cgg ctg gca gga att gag gag cgc tg - #c aag gag atg gca gag     2801     Lys Gly Arg Leu Ala Gly Ile Glu Glu Arg Cy - #s Lys Glu Met Ala Glu     #       820     - tta aca cgg cta agg aag cag gct ctg cag ga - #c acc ctg gcc ctg tac     2849     Leu Thr Arg Leu Arg Lys Gln Ala Leu Gln As - #p Thr Leu Ala Leu Tyr     #   835     - aag atg ttc agt gag gct gat gcc tgt gag ct - #c tgg att gac gag aag     2897     Lys Met Phe Ser Glu Ala Asp Ala Cys Glu Le - #u Trp Ile Asp Glu Lys     840                 8 - #45                 8 - #50                 8 -     #55     - gag cag tgg ctc aac aac atg cag atc cca ga - #g aag ctg gag gac ctg     2945     Glu Gln Trp Leu Asn Asn Met Gln Ile Pro Gl - #u Lys Leu Glu Asp Leu     #               870     - gaa gtc atc cag cac aga ttt gag agc cta ga - #a cca gaa atg aac aac     2993     Glu Val Ile Gln His Arg Phe Glu Ser Leu Gl - #u Pro Glu Met Asn Asn     #           885     - cag gct tcc cgg gtt gct gtg gtg aac cag at - #t gca cgg cag ctg atg     3041     Gln Ala Ser Arg Val Ala Val Val Asn Gln Il - #e Ala Arg Gln Leu Met     #       900     - cac aat ggc cac ccc agt gaa aag gaa atc ag - #a gct cag caa gac aaa     3089     His Asn Gly His Pro Ser Glu Lys Glu Ile Ar - #g Ala Gln Gln Asp Lys     #   915     - ctc aac acg agg tgg agt cag ttc aga gaa ct - #g gtg gac agg aaa aag     3137     Leu Asn Thr Arg Trp Ser Gln Phe Arg Glu Le - #u Val Asp Arg Lys Lys     920                 9 - #25                 9 - #30                 9 -     #35     - gat gct ctt ctg tct gcc ctg agc atc cag aa - #c tac cac ctc gag tgc     3185     Asp Ala Leu Leu Ser Ala Leu Ser Ile Gln As - #n Tyr His Leu Glu Cys     #               950     - aat gaa acc aaa tcc tgc atc cgg gag aag ac - #c aag gtc atc gag tct     3233     Asn Glu Thr Lys Ser Cys Ile Arg Glu Lys Th - #r Lys Val Ile Glu Ser     #           965     - acc caa gac ctt ggc aat gac ctg gca ggt gt - #c atg gcc ctg cag tgc     3281     Thr Gln Asp Leu Gly Asn Asp Leu Ala Gly Va - #l Met Ala Leu Gln Cys     #       980     - aag ctg act ggc atg gaa cga gac ttg gta gc - #c att gag gcg aag ctg     3329     Lys Leu Thr Gly Met Glu Arg Asp Leu Val Al - #a Ile Glu Ala Lys Leu     #   995     - agt gac ctg cag aaa gaa gct gag aag ctg ga - #g tcc gag cac cct gac     3377     Ser Asp Leu Gln Lys Glu Ala Glu Lys Leu Gl - #u Ser Glu His Pro Asp     #              10150  5     - cag gct caa gct atc ctg tct cgg ctg gcc ga - #g atc agt gat gtg tgg     3425     Gln Ala Gln Ala Ile Leu Ser Arg Leu Ala Gl - #u Ile Ser Asp Val Trp     #             10305     - gag gaa atg aag aca acc ctg aag aac cga ga - #g gcc tcc ctg gga gag     3473     Glu Glu Met Lys Thr Thr Leu Lys Asn Arg Gl - #u Ala Ser Leu Gly Glu     #         10450     - gcc agc aag ctg cag cag ttt ctg cgg gac tt - #g gac gac ttc cag tct     3521     Ala Ser Lys Leu Gln Gln Phe Leu Arg Asp Le - #u Asp Asp Phe Gln Ser     #     10605     - tgg ctc tcc agg acc cag act gct atc gcc tc - #a gag gac atg ccc aat     3569     Trp Leu Ser Arg Thr Gln Thr Ala Ile Ala Se - #r Glu Asp Met Pro Asn     # 10750     - acc ctc act gag gca gag aag ctt ctc aca ca - #g cac gag aat atc aaa     3617     Thr Leu Thr Glu Ala Glu Lys Leu Leu Thr Gl - #n His Glu Asn Ile Lys     #              10950  5     - aat gag atc gac aat tat gag gaa gac tac ca - #g aag atg cgg gac atg     3665     Asn Glu Ile Asp Asn Tyr Glu Glu Asp Tyr Gl - #n Lys Met Arg Asp Met     #             11105     - ggc gag atg gtc acc cag ggg cag act gat gc - #c cag tat atg ttt ctg     3713     Gly Glu Met Val Thr Gln Gly Gln Thr Asp Al - #a Gln Tyr Met Phe Leu     #         11250     - cgg cag cgg ctg cag gcc tta gac act ggc tg - #g aat gag ctc cac aaa     3761     Arg Gln Arg Leu Gln Ala Leu Asp Thr Gly Tr - #p Asn Glu Leu His Lys     #     11405     - atg tgg gag aac agg caa aac ctc ctc tcc ca - #g tcc cat gcc tac cag     3809     Met Trp Glu Asn Arg Gln Asn Leu Leu Ser Gl - #n Ser His Ala Tyr Gln     # 11550     - cag ttc ctt agg gac acc aaa caa gct gaa gc - #t ttt ctt aat aac cag     3857     Gln Phe Leu Arg Asp Thr Lys Gln Ala Glu Al - #a Phe Leu Asn Asn Gln     #              11750  5     - gag tat gtt ttg gct cat act gaa atg ccc ac - #c acc ctg gaa gga gct     3905     Glu Tyr Val Leu Ala His Thr Glu Met Pro Th - #r Thr Leu Glu Gly Ala     #             11905     - gaa gca gcc att aaa aag cag gag gac ttc at - #g acc acc atg gat gcc     3953     Glu Ala Ala Ile Lys Lys Gln Glu Asp Phe Me - #t Thr Thr Met Asp Ala     #         12050     - aac gag gag aag atc aat gct gtt gtg gag ac - #t ggc cga aga ctg gtg     4001     Asn Glu Glu Lys Ile Asn Ala Val Val Glu Th - #r Gly Arg Arg Leu Val     #     12205     - agc gat ggg aac atc aac tcc gac cgc atc ca - #g gag aag gtg gac tct     4049     Ser Asp Gly Asn Ile Asn Ser Asp Arg Ile Gl - #n Glu Lys Val Asp Ser     # 12350     - att gac gac aga cac agg aag aat cga gaa gc - #a gcc agt gaa ctt ctg     4097     Ile Asp Asp Arg His Arg Lys Asn Arg Glu Al - #a Ala Ser Glu Leu Leu     #              12550  5     - atg agg tta aag gac aac cgt gat cta cag aa - #g ttc ctg caa gat tgt     4145     Met Arg Leu Lys Asp Asn Arg Asp Leu Gln Ly - #s Phe Leu Gln Asp Cys     #             12705     - caa gag ctg tcc ctc tgg atc aat gaa aag at - #g ctt aca gct caa gac     4193     Gln Glu Leu Ser Leu Trp Ile Asn Glu Lys Me - #t Leu Thr Ala Gln Asp     #         12850     - atg tct tat gat gaa gcc aga aat ctg cac ag - #t aaa tgg tta aag cat     4241     Met Ser Tyr Asp Glu Ala Arg Asn Leu His Se - #r Lys Trp Leu Lys His     #     13005     - caa gca ttt atg gcg gaa ctt gca tcc aac aa - #a gaa tgg ctt gac aaa     4289     Gln Ala Phe Met Ala Glu Leu Ala Ser Asn Ly - #s Glu Trp Leu Asp Lys     # 13150     - att gag aag gaa gga atg cag ctt att tca ga - #a aag cca gaa aca gaa     4337     Ile Glu Lys Glu Gly Met Gln Leu Ile Ser Gl - #u Lys Pro Glu Thr Glu     #              13350  5     - gct gtg gta aag gaa aaa ctc act ggt tta ca - #t aaa atg tgg gaa gtc     4385     Ala Val Val Lys Glu Lys Leu Thr Gly Leu Hi - #s Lys Met Trp Glu Val     #             13505     - ctt gaa tcc aca acc cag acc aag gcc cag cg - #g ctc ttt gat gca aat     4433     Leu Glu Ser Thr Thr Gln Thr Lys Ala Gln Ar - #g Leu Phe Asp Ala Asn     #         13650     - aag gct gag ctt ttc aca caa agc tgc gca ga - #t ctt gac aaa tgg cta     4481     Lys Ala Glu Leu Phe Thr Gln Ser Cys Ala As - #p Leu Asp Lys Trp Leu     #     13805     - cat ggc ctg gag agc cag att caa tct gac ga - #c tat ggc aaa gac ctt     4529     His Gly Leu Glu Ser Gln Ile Gln Ser Asp As - #p Tyr Gly Lys Asp Leu     # 13950     - acc agt gtc aat att ctt ctg aaa aag caa ca - #g atg ctg gag aat cag     4577     Thr Ser Val Asn Ile Leu Leu Lys Lys Gln Gl - #n Met Leu Glu Asn Gln     #              14150  5     - atg gaa gtt cgg aag aaa gag atc gag gaa ct - #g cag agc caa gcc cag     4625     Met Glu Val Arg Lys Lys Glu Ile Glu Glu Le - #u Gln Ser Gln Ala Gln     #             14305     - gcg ctg agt cag gag ggg aag agc aca gat ga - #g gtg gac agc aaa cgc     4673     Ala Leu Ser Gln Glu Gly Lys Ser Thr Asp Gl - #u Val Asp Ser Lys Arg     #         14450     - ctt act gtg cag acc aag ttc atg gag ctt ct - #g gag ccc ttg agt gag     4721     Leu Thr Val Gln Thr Lys Phe Met Glu Leu Le - #u Glu Pro Leu Ser Glu     #     14605     - agg aag cat aac ctg tta gct tcc aag gag at - #c cat cag ttc aac agg     4769     Arg Lys His Asn Leu Leu Ala Ser Lys Glu Il - #e His Gln Phe Asn Arg     # 14750     - gat gtg gag gac gaa atc cta tgg gtt ggc ga - #g agg atg cct ttg gca     4817     Asp Val Glu Asp Glu Ile Leu Trp Val Gly Gl - #u Arg Met Pro Leu Ala     #              14950  5     - act tcc aca gat cat ggc cat aac ctt caa ac - #t gtg cag ctg tta ata     4865     Thr Ser Thr Asp His Gly His Asn Leu Gln Th - #r Val Gln Leu Leu Ile     #             15105     - aag aaa aac cag acc ctc cag aaa gaa atc ca - #g gga cac cag cct cgt     4913     Lys Lys Asn Gln Thr Leu Gln Lys Glu Ile Gl - #n Gly His Gln Pro Arg     #         15250     - att gat gac atc ttt gag agg agt caa aac at - #c atc aca gat agc agc     4961     Ile Asp Asp Ile Phe Glu Arg Ser Gln Asn Il - #e Ile Thr Asp Ser Ser     #     15405     - agc ctc aat gcc gag gct atc agg cag agg ct - #c gct gac ctg aag cag     5009     Ser Leu Asn Ala Glu Ala Ile Arg Gln Arg Le - #u Ala Asp Leu Lys Gln     # 15550     - ctg tgg ggg ctc ctc att gag gaa act gag aa - #a cgc cat aga cgg ctg     5057     Leu Trp Gly Leu Leu Ile Glu Glu Thr Glu Ly - #s Arg His Arg Arg Leu     #              15750  5     - gag gag gca cac aag gcg cag cag tac tac tt - #t gat gca gct gaa gcc     5105     Glu Glu Ala His Lys Ala Gln Gln Tyr Tyr Ph - #e Asp Ala Ala Glu Ala     #             15905     - gag gca tgg atg agt gaa cag gag ttg tac at - #g atg tct gag gaa aag     5153     Glu Ala Trp Met Ser Glu Gln Glu Leu Tyr Me - #t Met Ser Glu Glu Lys     #         16050     - gcc aag gat gag cag agt gct gtc tct atg tt - #g aaa aag cac cag att     5201     Ala Lys Asp Glu Gln Ser Ala Val Ser Met Le - #u Lys Lys His Gln Ile     #     16205     - tta gag caa gct gtt gag gac tat gca gag ac - #a gta cac cag ctc tcc     5249     Leu Glu Gln Ala Val Glu Asp Tyr Ala Glu Th - #r Val His Gln Leu Ser     # 16350     - aag act agc cgg gcg ctg gtg gct gac agc ca - #t ccc gaa agt gag cgt     5297     Lys Thr Ser Arg Ala Leu Val Ala Asp Ser Hi - #s Pro Glu Ser Glu Arg     #              16550  5     - att agc atg cgg cag tca aag gtc gac aag ct - #g tat gct ggc ctg aag     5345     Ile Ser Met Arg Gln Ser Lys Val Asp Lys Le - #u Tyr Ala Gly Leu Lys     #             16705     - gac ctt gct gag gag agg aga gga aaa ctt ga - #t gag agg cac agg ctg     5393     Asp Leu Ala Glu Glu Arg Arg Gly Lys Leu As - #p Glu Arg His Arg Leu     #         16850     - ttc cag ctc aac aga gag gtg gat gac ctg ga - #a cag tgg atc gct gag     5441     Phe Gln Leu Asn Arg Glu Val Asp Asp Leu Gl - #u Gln Trp Ile Ala Glu     #     17005     - agg gaa gtg gtc gca ggc tcc cat gag ttg gg - #a cag gac tat gag cat     5489     Arg Glu Val Val Ala Gly Ser His Glu Leu Gl - #y Gln Asp Tyr Glu His     # 17150     - gtc acg atg tta caa gaa cgg ttc cga gaa tt - #t gct cga gac aca gga     5537     Val Thr Met Leu Gln Glu Arg Phe Arg Glu Ph - #e Ala Arg Asp Thr Gly     #              17350  5     - aac att ggg cag gag cgt gtg gat aca gtt aa - #t aac atg gca gat gaa     5585     Asn Ile Gly Gln Glu Arg Val Asp Thr Val As - #n Asn Met Ala Asp Glu     #             17505     - ctc atc aac tct gga cat tca gat gct gcc ac - #c att gct gag tgg aaa     5633     Leu Ile Asn Ser Gly His Ser Asp Ala Ala Th - #r Ile Ala Glu Trp Lys     #         17650     - gat ggt ctc aat gaa gcc tgg gct gac ctc ct - #g gag ctc att gac aca     5681     Asp Gly Leu Asn Glu Ala Trp Ala Asp Leu Le - #u Glu Leu Ile Asp Thr     #     17805     - aga aca cag att ctt gct gcc tca tat gaa ct - #t cat aag ttt tac cat     5729     Arg Thr Gln Ile Leu Ala Ala Ser Tyr Glu Le - #u His Lys Phe Tyr His     # 17950     - gat gcc aag gag atc ttt ggc cga atc cag ga - #c aaa cac aag aaa ctc     5777     Asp Ala Lys Glu Ile Phe Gly Arg Ile Gln As - #p Lys His Lys Lys Leu     #              18150  5     - cct gag gag ctt gga aga gat caa aac act gt - #g gaa act tta cag aga     5825     Pro Glu Glu Leu Gly Arg Asp Gln Asn Thr Va - #l Glu Thr Leu Gln Arg     #             18305     - atg cac acc acc ttt gag cac gac atc caa gc - #t ctg ggc act cag gtg     5873     Met His Thr Thr Phe Glu His Asp Ile Gln Al - #a Leu Gly Thr Gln Val     #         18450     - agg cag ctg cag gag gat gca gct cgc ctc ca - #g gca gcc tat gca ggg     5921     Arg Gln Leu Gln Glu Asp Ala Ala Arg Leu Gl - #n Ala Ala Tyr Ala Gly     #     18605     - gac aag gct gat gac atc cag aag cgt gag aa - #t gag gtc ctg gaa gcc     5969     Asp Lys Ala Asp Asp Ile Gln Lys Arg Glu As - #n Glu Val Leu Glu Ala     # 18750     - tgg aag tcc ctg ctg gat gct tgt gag ggt cg - #c agg gtg cgg ctg gta     6017     Trp Lys Ser Leu Leu Asp Ala Cys Glu Gly Ar - #g Arg Val Arg Leu Val     #              18950  5     - gac aca gga gac aag ttc cgc ttc ttc agc at - #g gtg cgt gac ctc atg     6065     Asp Thr Gly Asp Lys Phe Arg Phe Phe Ser Me - #t Val Arg Asp Leu Met     #             19105     - ctc tgg atg gaa gat gtc atc cgg cag atc ga - #g gcc cag gag aaa cca     6113     Leu Trp Met Glu Asp Val Ile Arg Gln Ile Gl - #u Ala Gln Glu Lys Pro     #         19250     - cgg gat gtg tca tct gtt gaa ctg tta atg aa - #t aat cat caa ggt atc     6161     Arg Asp Val Ser Ser Val Glu Leu Leu Met As - #n Asn His Gln Gly Ile     #     19405     - aaa gct gaa att gat gct cgt aat gac agc tt - #t aca gcc tgc att gag     6209     Lys Ala Glu Ile Asp Ala Arg Asn Asp Ser Ph - #e Thr Ala Cys Ile Glu     # 19550     - ctt ggg aaa tcc ctg ctg gca cgg aaa cac ta - #t gct tct gag gag atc     6257     Leu Gly Lys Ser Leu Leu Ala Arg Lys His Ty - #r Ala Ser Glu Glu Ile     #              19750  5     - aag gaa aag tta ctg cag ctg aca gag aaa ag - #a aaa gaa atg att gac     6305     Lys Glu Lys Leu Leu Gln Leu Thr Glu Lys Ar - #g Lys Glu Met Ile Asp     #             19905     - aag tgg gaa gac cgg tgg gag tgg tta aga ct - #g att ttg gag gtc cat     6353     Lys Trp Glu Asp Arg Trp Glu Trp Leu Arg Le - #u Ile Leu Glu Val His     #         20050     - cag ttc tca agg gat gcc agt gtg gca gag gc - #t tgg ctg ctt gga cag     6401     Gln Phe Ser Arg Asp Ala Ser Val Ala Glu Al - #a Trp Leu Leu Gly Gln     #     20205     - gaa cca tac cta tcc agc cgt gaa att ggc ca - #g agt gta gac gaa gtg     6449     Glu Pro Tyr Leu Ser Ser Arg Glu Ile Gly Gl - #n Ser Val Asp Glu Val     # 20350     - gag aag ctt att aag cgc cat gag gcg ttt ga - #a aag tct gca gcg acc     6497     Glu Lys Leu Ile Lys Arg His Glu Ala Phe Gl - #u Lys Ser Ala Ala Thr     #              20550  5     - tgg gat gag aga ttc tct gct ctg gaa agg ct - #g aca acg ttg gag cta     6545     Trp Asp Glu Arg Phe Ser Ala Leu Glu Arg Le - #u Thr Thr Leu Glu Leu     #             20705     - ctg gaa gtg cgc aga cag caa gag gaa gaa ga - #a aga aag agg cgg cca     6593     Leu Glu Val Arg Arg Gln Gln Glu Glu Glu Gl - #u Arg Lys Arg Arg Pro     #         20850     - cct tct ccg gac cca aac acg aag gtt tca ga - #g gag gct gag tcc cag     6641     Pro Ser Pro Asp Pro Asn Thr Lys Val Ser Gl - #u Glu Ala Glu Ser Gln     #     21005     - caa tgg gat act tca aaa gga gac caa gtt tc - #c cag aat ggt ttg ccg     6689     Gln Trp Asp Thr Ser Lys Gly Asp Gln Val Se - #r Gln Asn Gly Leu Pro     # 21150     - gct gag cag gga tct cca cgg gtt agt tac cg - #c tct caa acg tac caa     6737     Ala Glu Gln Gly Ser Pro Arg Val Ser Tyr Ar - #g Ser Gln Thr Tyr Gln     #              21350  5     - aac tac aaa aac ttt aat agc aga cgg aca gc - #c agt gac cat tca tgg     6785     Asn Tyr Lys Asn Phe Asn Ser Arg Arg Thr Al - #a Ser Asp His Ser Trp     #             21505     - tct gga atg tgaagttcac taccatttgt caagaaccac tctgtccac - #a     6834     Ser Gly Met     - tcctttgacc ttttggcttc cacgtcaccc agagtgttaa aatttttact ta - #attcatag     6894     - ctgtccttga tttcatattt gtttgcattt aatttatgtt tctttggatc ct - #cattgcct     6954     #         6960     - <210> SEQ ID NO 4     <211> LENGTH: 2154     <212> TYPE: PRT     <213> ORGANISM: Mus musculus     - <400> SEQUENCE: 4     - Met Glu Leu Gln Arg Thr Ser Ser Val Ser Gl - #y Pro Leu Ser Pro Ala     #                 15     - Tyr Thr Gly Gln Val Pro Tyr Asn Tyr Asn Gl - #n Leu Glu Gly Arg Phe     #             30     - Lys Gln Leu Gln Asp Glu Arg Glu Ala Val Gl - #n Lys Lys Thr Phe Thr     #         45     - Lys Trp Val Asn Ser His Leu Ala Arg Val Se - #r Cys Arg Ile Thr Asp     #     60     - Leu Tyr Thr Asp Leu Arg Asp Gly Arg Met Le - #u Ile Lys Leu Leu Glu     # 80     - Val Leu Ser Gly Glu Arg Leu Pro Lys Pro Th - #r Lys Gly Arg Met Arg     #                 95     - Ile His Cys Leu Glu Asn Val Asp Lys Ala Le - #u Gln Phe Leu Lys Glu     #           110     - Gln Arg Val His Leu Glu Asn Met Gly Ser Hi - #s Asp Ile Val Asp Gly     #       125     - Asn His Arg Leu Thr Leu Gly Leu Ile Trp Th - #r Ile Ile Leu Arg Phe     #   140     - Gln Ile Gln Asp Ile Ser Val Glu Thr Glu As - #p Asn Lys Glu Lys Lys     145                 1 - #50                 1 - #55                 1 -     #60     - Ser Ala Lys Asp Ala Leu Leu Leu Trp Cys Gl - #n Met Lys Thr Ala Gly     #               175     - Tyr Pro Asn Val Asn Ile His Asn Phe Thr Th - #r Ser Trp Arg Asp Gly     #           190     - Met Ala Phe Asn Ala Leu Ile His Lys His Ar - #g Pro Asp Leu Ile Asp     #       205     - Phe Asp Lys Leu Lys Lys Ser Asn Ala His Ty - #r Asn Leu Gln Asn Ala     #   220     - Phe Asn Leu Ala Glu Gln His Leu Gly Leu Th - #r Lys Leu Leu Asp Pro     225                 2 - #30                 2 - #35                 2 -     #40     - Glu Asp Ile Ser Val Asp His Pro Asp Glu Ly - #s Ser Ile Ile Thr Tyr     #               255     - Val Val Thr Tyr Tyr His Tyr Phe Ser Lys Me - #t Lys Ala Leu Ala Val     #           270     - Glu Gly Lys Arg Ile Gly Lys Val Leu Asp As - #n Ala Ile Glu Thr Glu     #       285     - Lys Met Ile Glu Lys Tyr Glu Thr Leu Ala Se - #r Asp Leu Leu Glu Trp     #   300     - Ile Glu Gln Thr Ile Ile Ile Leu Asn Asn Ar - #g Lys Phe Ala Asn Ser     305                 3 - #10                 3 - #15                 3 -     #20     - Leu Val Gly Val Gln Gln Gln Leu Gln Ala Ph - #e Asn Thr Tyr Arg Thr     #               335     - Val Glu Lys Pro Pro Lys Phe Thr Glu Lys Gl - #y Asn Leu Glu Val Leu     #           350     - Leu Phe Ala Ile Gln Ser Lys Met Arg Ala As - #n Asn Gln Lys Val Tyr     #       365     - Met Pro Arg Glu Gly Lys Leu Ile Ser Asp Il - #e Asn Lys Ala Trp Glu     #   380     - Arg Leu Glu Lys Ala Glu His Glu Arg Glu Le - #u Ala Leu Arg Asn Glu     385                 3 - #90                 3 - #95                 4 -     #00     - Leu Ile Arg Gln Glu Lys Leu Glu Gln Leu Al - #a Arg Arg Phe Asp Arg     #               415     - Lys Ala Ala Met Arg Glu Thr Trp Leu Ser Gl - #u Asn Gln Arg Leu Val     #           430     - Ser Gln Asp Asn Phe Gly Phe Asp Leu Pro Al - #a Val Glu Ala Ala Thr     #       445     - Lys Lys His Glu Ala Ile Glu Thr Asp Ile Al - #a Ala Tyr Glu Glu Arg     #   460     - Val Gln Ala Val Val Ala Val Ala Arg Glu Le - #u Glu Ala Glu Asn Tyr     465                 4 - #70                 4 - #75                 4 -     #80     - His Asp Ile Lys Arg Ile Thr Ala Arg Lys As - #p Asn Val Ile Arg Leu     #               495     - Trp Glu Tyr Leu Leu Glu Leu Leu Arg Ala Ar - #g Arg Gln Arg Leu Glu     #           510     - Met Asn Leu Gly Leu Gln Lys Ile Phe Gln Gl - #u Met Leu Tyr Ile Met     #       525     - Asp Trp Met Asp Glu Met Lys Val Leu Leu Le - #u Ser Gln Asp Tyr Gly     #   540     - Lys His Leu Leu Gly Val Glu Asp Leu Leu Gl - #n Lys His Ala Leu Val     545                 5 - #50                 5 - #55                 5 -     #60     - Glu Ala Asp Ile Ala Ile Gln Ala Glu Arg Va - #l Arg Gly Val Asn Ala     #               575     - Ser Ala Gln Lys Phe Ala Thr Asp Gly Glu Gl - #y Tyr Lys Pro Cys Asp     #           590     - Pro Gln Val Ile Arg Asp Arg Val Ala His Me - #t Glu Phe Cys Tyr Gln     #       605     - Glu Leu Cys Gln Leu Ala Ala Glu Arg Arg Al - #a Arg Leu Glu Glu Ser     #   620     - Arg Arg Leu Trp Lys Phe Phe Trp Glu Met Al - #a Glu Glu Glu Gly Trp     625                 6 - #30                 6 - #35                 6 -     #40     - Ile Arg Glu Lys Glu Lys Ile Leu Ser Ser As - #p Asp Tyr Gly Lys Asp     #               655     - Leu Thr Ser Val Met Arg Leu Leu Ser Lys Hi - #s Arg Ala Phe Glu Asp     #           670     - Glu Met Ser Gly Arg Ser Gly His Phe Glu Gl - #n Ala Ile Lys Glu Gly     #       685     - Glu Asp Met Ile Ala Glu Glu His Phe Gly Se - #r Glu Lys Ile Arg Glu     #   700     - Arg Ile Ile Tyr Ile Arg Glu Gln Trp Ala As - #n Leu Glu Gln Leu Ser     705                 7 - #10                 7 - #15                 7 -     #20     - Ala Ile Arg Lys Lys Arg Leu Glu Glu Ala Se - #r Leu Leu His Gln Phe     #               735     - Gln Ala Asp Ala Asp Asp Ile Asp Ala Trp Me - #t Leu Asp Ile Leu Lys     #           750     - Ile Val Ser Ser Asn Asp Val Gly His Asp Gl - #u Tyr Ser Thr Gln Ser     #       765     - Leu Val Lys Lys His Lys Asp Val Ala Glu Gl - #u Ile Thr Asn Cys Arg     #   780     - Pro Thr Ile Asp Thr Leu His Glu Gln Ala Se - #r Ala Leu Pro Gln Ala     785                 7 - #90                 7 - #95                 8 -     #00     - His Ala Glu Ser Pro Asp Val Lys Gly Arg Le - #u Ala Gly Ile Glu Glu     #               815     - Arg Cys Lys Glu Met Ala Glu Leu Thr Arg Le - #u Arg Lys Gln Ala Leu     #           830     - Gln Asp Thr Leu Ala Leu Tyr Lys Met Phe Se - #r Glu Ala Asp Ala Cys     #       845     - Glu Leu Trp Ile Asp Glu Lys Glu Gln Trp Le - #u Asn Asn Met Gln Ile     #   860     - Pro Glu Lys Leu Glu Asp Leu Glu Val Ile Gl - #n His Arg Phe Glu Ser     865                 8 - #70                 8 - #75                 8 -     #80     - Leu Glu Pro Glu Met Asn Asn Gln Ala Ser Ar - #g Val Ala Val Val Asn     #               895     - Gln Ile Ala Arg Gln Leu Met His Asn Gly Hi - #s Pro Ser Glu Lys Glu     #           910     - Ile Arg Ala Gln Gln Asp Lys Leu Asn Thr Ar - #g Trp Ser Gln Phe Arg     #       925     - Glu Leu Val Asp Arg Lys Lys Asp Ala Leu Le - #u Ser Ala Leu Ser Ile     #   940     - Gln Asn Tyr His Leu Glu Cys Asn Glu Thr Ly - #s Ser Cys Ile Arg Glu     945                 9 - #50                 9 - #55                 9 -     #60     - Lys Thr Lys Val Ile Glu Ser Thr Gln Asp Le - #u Gly Asn Asp Leu Ala     #               975     - Gly Val Met Ala Leu Gln Cys Lys Leu Thr Gl - #y Met Glu Arg Asp Leu     #           990     - Val Ala Ile Glu Ala Lys Leu Ser Asp Leu Gl - #n Lys Glu Ala Glu Lys     #     100500     - Leu Glu Ser Glu His Pro Asp Gln Ala Gln Al - #a Ile Leu Ser Arg Leu     # 10205     - Ala Glu Ile Ser Asp Val Trp Glu Glu Met Ly - #s Thr Thr Leu Lys Asn     #              10405  0     - Arg Glu Ala Ser Leu Gly Glu Ala Ser Lys Le - #u Gln Gln Phe Leu Arg     #             10550     - Asp Leu Asp Asp Phe Gln Ser Trp Leu Ser Ar - #g Thr Gln Thr Ala Ile     #         10705     - Ala Ser Glu Asp Met Pro Asn Thr Leu Thr Gl - #u Ala Glu Lys Leu Leu     #     108580     - Thr Gln His Glu Asn Ile Lys Asn Glu Ile As - #p Asn Tyr Glu Glu Asp     # 11005     - Tyr Gln Lys Met Arg Asp Met Gly Glu Met Va - #l Thr Gln Gly Gln Thr     #              11205  0     - Asp Ala Gln Tyr Met Phe Leu Arg Gln Arg Le - #u Gln Ala Leu Asp Thr     #             11350     - Gly Trp Asn Glu Leu His Lys Met Trp Glu As - #n Arg Gln Asn Leu Leu     #         11505     - Ser Gln Ser His Ala Tyr Gln Gln Phe Leu Ar - #g Asp Thr Lys Gln Ala     #     116560     - Glu Ala Phe Leu Asn Asn Gln Glu Tyr Val Le - #u Ala His Thr Glu Met     # 11805     - Pro Thr Thr Leu Glu Gly Ala Glu Ala Ala Il - #e Lys Lys Gln Glu Asp     #              12005  0     - Phe Met Thr Thr Met Asp Ala Asn Glu Glu Ly - #s Ile Asn Ala Val Val     #             12150     - Glu Thr Gly Arg Arg Leu Val Ser Asp Gly As - #n Ile Asn Ser Asp Arg     #         12305     - Ile Gln Glu Lys Val Asp Ser Ile Asp Asp Ar - #g His Arg Lys Asn Arg     #     124540     - Glu Ala Ala Ser Glu Leu Leu Met Arg Leu Ly - #s Asp Asn Arg Asp Leu     # 12605     - Gln Lys Phe Leu Gln Asp Cys Gln Glu Leu Se - #r Leu Trp Ile Asn Glu     #              12805  0     - Lys Met Leu Thr Ala Gln Asp Met Ser Tyr As - #p Glu Ala Arg Asn Leu     #             12950     - His Ser Lys Trp Leu Lys His Gln Ala Phe Me - #t Ala Glu Leu Ala Ser     #         13105     - Asn Lys Glu Trp Leu Asp Lys Ile Glu Lys Gl - #u Gly Met Gln Leu Ile     #     132520     - Ser Glu Lys Pro Glu Thr Glu Ala Val Val Ly - #s Glu Lys Leu Thr Gly     # 13405     - Leu His Lys Met Trp Glu Val Leu Glu Ser Th - #r Thr Gln Thr Lys Ala     #              13605  0     - Gln Arg Leu Phe Asp Ala Asn Lys Ala Glu Le - #u Phe Thr Gln Ser Cys     #             13750     - Ala Asp Leu Asp Lys Trp Leu His Gly Leu Gl - #u Ser Gln Ile Gln Ser     #         13905     - Asp Asp Tyr Gly Lys Asp Leu Thr Ser Val As - #n Ile Leu Leu Lys Lys     #     140500     - Gln Gln Met Leu Glu Asn Gln Met Glu Val Ar - #g Lys Lys Glu Ile Glu     # 14205     - Glu Leu Gln Ser Gln Ala Gln Ala Leu Ser Gl - #n Glu Gly Lys Ser Thr     #              14405  0     - Asp Glu Val Asp Ser Lys Arg Leu Thr Val Gl - #n Thr Lys Phe Met Glu     #             14550     - Leu Leu Glu Pro Leu Ser Glu Arg Lys His As - #n Leu Leu Ala Ser Lys     #         14705     - Glu Ile His Gln Phe Asn Arg Asp Val Glu As - #p Glu Ile Leu Trp Val     #     148580     - Gly Glu Arg Met Pro Leu Ala Thr Ser Thr As - #p His Gly His Asn Leu     # 15005     - Gln Thr Val Gln Leu Leu Ile Lys Lys Asn Gl - #n Thr Leu Gln Lys Glu     #              15205  0     - Ile Gln Gly His Gln Pro Arg Ile Asp Asp Il - #e Phe Glu Arg Ser Gln     #             15350     - Asn Ile Ile Thr Asp Ser Ser Ser Leu Asn Al - #a Glu Ala Ile Arg Gln     #         15505     - Arg Leu Ala Asp Leu Lys Gln Leu Trp Gly Le - #u Leu Ile Glu Glu Thr     #     156560     - Glu Lys Arg His Arg Arg Leu Glu Glu Ala Hi - #s Lys Ala Gln Gln Tyr     # 15805     - Tyr Phe Asp Ala Ala Glu Ala Glu Ala Trp Me - #t Ser Glu Gln Glu Leu     #              16005  0     - Tyr Met Met Ser Glu Glu Lys Ala Lys Asp Gl - #u Gln Ser Ala Val Ser     #             16150     - Met Leu Lys Lys His Gln Ile Leu Glu Gln Al - #a Val Glu Asp Tyr Ala     #         16305     - Glu Thr Val His Gln Leu Ser Lys Thr Ser Ar - #g Ala Leu Val Ala Asp     #     164540     - Ser His Pro Glu Ser Glu Arg Ile Ser Met Ar - #g Gln Ser Lys Val Asp     # 16605     - Lys Leu Tyr Ala Gly Leu Lys Asp Leu Ala Gl - #u Glu Arg Arg Gly Lys     #              16805  0     - Leu Asp Glu Arg His Arg Leu Phe Gln Leu As - #n Arg Glu Val Asp Asp     #             16950     - Leu Glu Gln Trp Ile Ala Glu Arg Glu Val Va - #l Ala Gly Ser His Glu     #         17105     - Leu Gly Gln Asp Tyr Glu His Val Thr Met Le - #u Gln Glu Arg Phe Arg     #     172520     - Glu Phe Ala Arg Asp Thr Gly Asn Ile Gly Gl - #n Glu Arg Val Asp Thr     # 17405     - Val Asn Asn Met Ala Asp Glu Leu Ile Asn Se - #r Gly His Ser Asp Ala     #              17605  0     - Ala Thr Ile Ala Glu Trp Lys Asp Gly Leu As - #n Glu Ala Trp Ala Asp     #             17750     - Leu Leu Glu Leu Ile Asp Thr Arg Thr Gln Il - #e Leu Ala Ala Ser Tyr     #         17905     - Glu Leu His Lys Phe Tyr His Asp Ala Lys Gl - #u Ile Phe Gly Arg Ile     #     180500     - Gln Asp Lys His Lys Lys Leu Pro Glu Glu Le - #u Gly Arg Asp Gln Asn     # 18205     - Thr Val Glu Thr Leu Gln Arg Met His Thr Th - #r Phe Glu His Asp Ile     #              18405  0     - Gln Ala Leu Gly Thr Gln Val Arg Gln Leu Gl - #n Glu Asp Ala Ala Arg     #             18550     - Leu Gln Ala Ala Tyr Ala Gly Asp Lys Ala As - #p Asp Ile Gln Lys Arg     #         18705     - Glu Asn Glu Val Leu Glu Ala Trp Lys Ser Le - #u Leu Asp Ala Cys Glu     #     188580     - Gly Arg Arg Val Arg Leu Val Asp Thr Gly As - #p Lys Phe Arg Phe Phe     # 19005     - Ser Met Val Arg Asp Leu Met Leu Trp Met Gl - #u Asp Val Ile Arg Gln     #              19205  0     - Ile Glu Ala Gln Glu Lys Pro Arg Asp Val Se - #r Ser Val Glu Leu Leu     #             19350     - Met Asn Asn His Gln Gly Ile Lys Ala Glu Il - #e Asp Ala Arg Asn Asp     #         19505     - Ser Phe Thr Ala Cys Ile Glu Leu Gly Lys Se - #r Leu Leu Ala Arg Lys     #     196560     - His Tyr Ala Ser Glu Glu Ile Lys Glu Lys Le - #u Leu Gln Leu Thr Glu     # 19805     - Lys Arg Lys Glu Met Ile Asp Lys Trp Glu As - #p Arg Trp Glu Trp Leu     #              20005  0     - Arg Leu Ile Leu Glu Val His Gln Phe Ser Ar - #g Asp Ala Ser Val Ala     #             20150     - Glu Ala Trp Leu Leu Gly Gln Glu Pro Tyr Le - #u Ser Ser Arg Glu Ile     #         20305     - Gly Gln Ser Val Asp Glu Val Glu Lys Leu Il - #e Lys Arg His Glu Ala     #     204540     - Phe Glu Lys Ser Ala Ala Thr Trp Asp Glu Ar - #g Phe Ser Ala Leu Glu     # 20605     - Arg Leu Thr Thr Leu Glu Leu Leu Glu Val Ar - #g Arg Gln Gln Glu Glu     #              20805  0     - Glu Glu Arg Lys Arg Arg Pro Pro Ser Pro As - #p Pro Asn Thr Lys Val     #             20950     - Ser Glu Glu Ala Glu Ser Gln Gln Trp Asp Th - #r Ser Lys Gly Asp Gln     #         21105     - Val Ser Gln Asn Gly Leu Pro Ala Glu Gln Gl - #y Ser Pro Arg Val Ser     #     212520     - Tyr Arg Ser Gln Thr Tyr Gln Asn Tyr Lys As - #n Phe Asn Ser Arg Arg     # 21405     - Thr Ala Ser Asp His Ser Trp Ser Gly Met     145                2150     - <210> SEQ ID NO 5     <211> LENGTH: 8176     <212> TYPE: DNA     <213> ORGANISM: Mus musculus     - <400> SEQUENCE: 5     - cctgcgtcct tcctcctttt cctccttccc tcctccctcc cgggtaattt at - #ttctagct       60     - tccaggcaag ggccacacaa ggaaggaaat ccacagggga ttagatgccg gg - #gtggtaac      120     - tccaccaggc taggttggac tctgcagcca acttcctatc agatcaccct gc - #acctattt      180     - ccgacccgac cggaatgcga ctggcttgag gtccagccct ttcgcctggg cg - #ggagcaga      240     - gccgcggaag ctgcttggag ttggatgggg gtaggaaggg gctggagcgg ga - #atcctacg      300     - atgcaactgg cctgggccta aggttgggca taatggagtt gcagaggaca tc - #cagcgttt      360     - cagggccgct gtcgccggcc tacaccgggc aggtgcctta caactacaac ca - #actggagg      420     - gaagattcaa acagctccaa gatgagcgtg aagctgtaca gaagaagacc tt - #caccaagt      480     - gggtcaattc ccaccttgca agagtgtcct gccgaatcac agacctgtac ac - #ggaccttc      540     - gagatggacg gatgctcatc aagctactgg aggtcctctc tggagagagg ct - #gcctaaac      600     - ccactaaggg acggatgcgg atccactgtc tggagaatgt cgacaaggct ct - #tcaattcc      660     - tgaaagagca gagagtccat cttgagaaca tgggctccca tgacattgtg ga - #tggaaacc      720     - accggctgac cctcggcctc atctggacaa ttattctgcg cttccagatc ca - #ggatatta      780     - gtgtggagac tgaagataac aaagagaaaa agtctgctaa ggatgcattg ct - #gctgtggt      840     - gccagatgaa gacagctggg taccccaatg tcaacattca caatttcacc ac - #tagctgga      900     - gggatggcat ggccttcaat gcactgatac ataaacatcg gcctgacctg at - #agattttg      960     - ataaactgaa gaaatctaat gcacactaca atctgcagaa tgcatttaac ct - #ggcagagc     1020     - agcaccttgg cctcactaaa ctgttagacc ctgaagatat cagtgtggac ca - #ccctgatg     1080     - agaagtctat catcacatac gtggtgactt actaccacta cttctccaag at - #gaaggcct     1140     - tggctgtcga aggaaagcgc attggaaagg tgcttgataa tgctatagaa ac - #agagaaaa     1200     - tgattgagaa gtacgagaca cttgcttctg accttctgga gtggattgaa ca - #aaccatca     1260     - tcatcctaaa caaccgcaaa tttgctaatt cactggttgg ggtccaacag ca - #gctccaag     1320     - cattcaacac gtaccgcaca gtggagaaac cacctaagtt tactgagaag gg - #gaatttgg     1380     - aggtgctcct tttcgcgatt cagagcaaga tgcgagcgaa taatcagaag gt - #ctacatgc     1440     - cccgcgaggg gaagctcatc tctgacatca acaaggcctg ggaaagactg ga - #aaaagcag     1500     - aacatgagag agaactggct ctgcggaatg agctcatacg gcaggaaaaa ct - #ggaacaac     1560     - tcgcccgaag atttgatcgc aaggcagcta tgagggagac atggctgagt ga - #aaaccagc     1620     - gtcttgtgtc tcaggacaac tttggatttg accttcccgc tgttgaggct gc - #taccaaaa     1680     - aacacgaggc cattgagaca gacatcgctg catatgaaga acgagttcag gc - #cgtggtgg     1740     - ctgtggccag ggaacttgaa gccgagaact accatgacat caagcgcatc ac - #agcgagga     1800     - aggacaatgt catccggctc tgggaatact tgctggaact gctcagggcc ag - #gaggcagc     1860     - gtcttgagat gaacctggga ttgcaaaaga tattccagga aatgctttat at - #tatggact     1920     - ggatggatga aatgaaggtg ctattgctgt ctcaagacta tggcaaacac tt - #acttggtg     1980     - ttgaagacct gttacagaag catgccctgg ttgaagcaga cattgcaatc ca - #agcagagc     2040     - gtgtaagagg tgtgaatgcc tctgcccaga agtttgcaac agatggggaa gg - #ctacaagc     2100     - catgtgaccc ccaggtaatt cgagaccgtg ttgcccacat ggagttctgc ta - #tcaagagc     2160     - tttgtcagct ggctgccgag cgtagggctc gcctggaaga gtcccgtcgc ct - #ctggaagt     2220     - tcttctggga gatggcagaa gaggaaggct ggatacgaga gaaggaaaag at - #cctgtcct     2280     - ctgatgatta cgggaaagac ttgaccagtg tcatgcgcct gctgagcaag ca - #ccgggcat     2340     - ttgaggatga gatgagtggc cgtagtggcc attttgagca ggccattaaa ga - #aggtgaag     2400     - acatgattgc agaggaacac tttggatcgg aaaagatccg tgagagaatc at - #ttatatcc     2460     - gggagcagtg ggccaacctg gaacagctct cagccattag gaagaagcgc ct - #agaggaag     2520     - cctcattact gcaccagttc caggctgatg ctgatgatat tgatgcttgg at - #gttagata     2580     - tactcaagat tgtctccagc aatgatgtgg gccatgatga gtactccacg ca - #gtctctgg     2640     - tcaagaagca taaagatgta gcagaagaga tcaccaactg caggcccact at - #tgacacac     2700     - tgcatgagca agccagtgcc cttccacaag cacatgcaga gtctccagat gt - #gaagggcc     2760     - ggctggcagg aattgaggag cgctgcaagg agatggcaga gttaacacgg ct - #aaggaagc     2820     - aggctctgca ggacaccctg gccctgtaca agatgttcag tgaggctgat gc - #ctgtgagc     2880     - tctggattga cgagaaggag cagtggctca acaacatgca gatcccagag aa - #gctggagg     2940     - acctggaagt catccagcac agatttgaga gcctagaacc agaaatgaac aa - #ccaggctt     3000     - cccgggttgc tgtggtgaac cagattgcac ggcagctgat gcacaatggc ca - #ccccagtg     3060     - aaaaggaaat cagagctcag caagacaaac tcaacacgag gtggagtcag tt - #cagagaac     3120     - tggtggacag gaaaaaggat gctcttctgt ctgccctgag catccagaac ta - #ccacctcg     3180     - agtgcaatga aaccaaatcc tgcatccggg agaagaccaa ggtcatcgag tc - #tacccaag     3240     - accttggcaa tgacctggca ggtgtcatgg ccctgcagtg caagctgact gg - #catggaac     3300     - gagacttggt agccattgag gcgaagctga gtgacctgca gaaagaagct ga - #gaagctgg     3360     - agtccgagca ccctgaccag gctcaagcta tcctgtctcg gctggccgag at - #cagtgatg     3420     - tgtgggagga aatgaagaca accctgaaga accgagaggc ctccctggga ga - #ggccagca     3480     - agctgcagca gtttctgcgg gacttggacg acttccagtc ttggctctcc ag - #gacccaga     3540     - ctgctatcgc ctcagaggac atgcccaata ccctcactga ggcagagaag ct - #tctcacac     3600     - agcacgagaa tatcaaaaat gagatcgaca attatgagga agactaccag aa - #gatgcggg     3660     - acatgggcga gatggtcacc caggggcaga ctgatgccca gtatatgttt ct - #gcggcagc     3720     - ggctgcaggc cttagacact ggctggaatg agctccacaa aatgtgggag aa - #caggcaaa     3780     - acctcctctc ccagtcccat gcctaccagc agttccttag ggacaccaaa ca - #agctgaag     3840     - cttttcttaa taaccaggag tatgttttgg ctcatactga aatgcccacc ac - #cctggaag     3900     - gagctgaagc agccattaaa aagcaggagg acttcatgac caccatggat gc - #caacgagg     3960     - agaagatcaa tgctgttgtg gagactggcc gaagactggt gagcgatggg aa - #catcaact     4020     - ccgaccgcat ccaggagaag gtggactcta ttgacgacag acacaggaag aa - #tcgagaag     4080     - cagccagtga acttctgatg aggttaaagg acaaccgtga tctacagaag tt - #cctgcaag     4140     - attgtcaaga gctgtccctc tggatcaatg aaaagatgct tacagctcaa ga - #catgtctt     4200     - atgatgaagc cagaaatctg cacagtaaat ggttaaagca tcaagcattt at - #ggcggaac     4260     - ttgcatccaa caaagaatgg cttgacaaaa ttgagaagga aggaatgcag ct - #tatttcag     4320     - aaaagccaga aacagaagct gtggtaaagg aaaaactcac tggtttacat aa - #aatgtggg     4380     - aagtccttga atccacaacc cagaccaagg cccagcggct ctttgatgca aa - #taaggctg     4440     - agcttttcac acaaagctgc gcagatcttg acaaatggct acatggcctg ga - #gagccaga     4500     - ttcaatctga cgactatggc aaagacctta ccagtgtcaa tattcttctg aa - #aaagcaac     4560     - agatgctgga gaatcagatg gaagttcgga agaaagagat cgaggaactg ca - #gagccaag     4620     - cccaggcgct gagtcaggag gggaagagca cagatgaggt ggacagcaaa cg - #ccttactg     4680     - tgcagaccaa gttcatggag cttctggagc ccttgagtga gaggaagcat aa - #cctgttag     4740     - cttccaagga gatccatcag ttcaacaggg atgtggagga cgaaatccta tg - #ggttggcg     4800     - agaggatgcc tttggcaact tccacagatc atggccataa ccttcaaact gt - #gcagctgt     4860     - taataaagaa aaaccagacc ctccagaaag aaatccaggg acaccagcct cg - #tattgatg     4920     - acatctttga gaggagtcaa aacatcatca cagatagcag cagcctcaat gc - #cgaggcta     4980     - tcaggcagag gctcgctgac ctgaagcagc tgtgggggct cctcattgag ga - #aactgaga     5040     - aacgccatag acggctggag gaggcacaca aggcgcagca gtactacttt ga - #tgcagctg     5100     - aagccgaggc atggatgagt gaacaggagt tgtacatgat gtctgaggaa aa - #ggccaagg     5160     - atgagcagag tgctgtctct atgttgaaaa agcaccagat tttagagcaa gc - #tgttgagg     5220     - actatgcaga gacagtacac cagctctcca agactagccg ggcgctggtg gc - #tgacagcc     5280     - atcccgaaag tgagcgtatt agcatgcggc agtcaaaggt cgacaagctg ta - #tgctggcc     5340     - tgaaggacct tgctgaggag aggagaggaa aacttgatga gaggcacagg ct - #gttccagc     5400     - tcaacagaga ggtggatgac ctggaacagt ggatcgctga gagggaagtg gt - #cgcaggct     5460     - cccatgagtt gggacaggac tatgagcatg tcacgatgtt acaagaacgg tt - #ccgagaat     5520     - ttgctcgaga cacaggaaac attgggcagg agcgtgtgga tacagttaat aa - #catggcag     5580     - atgaactcat caactctgga cattcagatg ctgccaccat tgctgagtgg aa - #agatggtc     5640     - tcaatgaagc ctgggctgac ctcctggagc tcattgacac aagaacacag at - #tcttgctg     5700     - cctcatatga acttcataag ttttaccatg atgccaagga gatctttggc cg - #aatccagg     5760     - acaaacacaa gaaactccct gaggagcttg gaagagatca aaacactgtg ga - #aactttac     5820     - agagaatgca caccaccttt gagcacgaca tccaagctct gggcactcag gt - #gaggcagc     5880     - tgcaggagga tgcagctcgc ctccaggcag cctatgcagg ggacaaggct ga - #tgacatcc     5940     - agaagcgtga gaatgaggtc ctggaagcct ggaagtccct gctggatgct tg - #tgagggtc     6000     - gcagggtgcg gctggtagac acaggagaca agttccgctt cttcagcatg gt - #gcgtgacc     6060     - tcatgctctg gatggaagat gtcatccggc agatcgaggc ccaggagaaa cc - #acgggatg     6120     - tgtcatctgt tgaactgtta atgaataatc atcaaggtat caaagctgaa at - #tgatgctc     6180     - gtaatgacag ctttacagcc tgcattgagc ttgggaaatc cctgctggca cg - #gaaacact     6240     - atgcttctga ggagatcaag gaaaagttac tgcagctgac agagaaaaga aa - #agaaatga     6300     - ttgacaagtg ggaagaccgg tgggagtggt taagactgat tttggaggtc ca - #tcagttct     6360     - caagggatgc cagtgtggca gaggcttggc tgcttggaca ggaaccatac ct - #atccagcc     6420     - gtgaaattgg ccagagtgta gacgaagtgg agaagcttat taagcgccat ga - #ggcgtttg     6480     - aaaagtctgc agcgacctgg gatgagagat tctctgctct ggaaaggctg ac - #aacgttgg     6540     - agctactgga agtgcgcaga cagcaagagg aagaagaaag aaagaggcgg cc - #accttctc     6600     - cggacccaaa cacgaaggtt tcagaggagg ctgagtccca gcaatgggat ac - #ttcaaaag     6660     - gagaccaagt ttcccagaat ggtttgccgg ctgagcaggg atctccacgg gt - #tagttacc     6720     - gctctcaaac gtaccaaaac tacaaaaact ttaatagcag acggacagcc ag - #tgaccatt     6780     - catggtctgg aatgtgaagt tcactaccat ttgtcaagaa ccactctgtc ca - #catccttt     6840     - gaccttttgg cttccacgtc acccagagtg ttaaaatttt tacttaattc at - #agctgtcc     6900     - ttgatttcat atttgtttgc atttaattta tgtttctttg gatcctcatt gc - #ctcaaagc     6960     - agcatactta atttttgttt atttattgtg agctttttac tttaagattt ta - #catgagta     7020     - atcaaaatta aattatagca taatgaaatt agactcttaa caggtacggc ac - #acacaagt     7080     - taatagtact ctgctatagg tgctatgtta cttacaagta ttattaacct at - #tggcttcc     7140     - attgtatagt agttagtaac tatgaaaact ggtttgtaag gaaggaaacg tt - #tactacta     7200     - aggttaggcc tgcagttgct ctggaacatt ccatggagaa tgcattcatc aa - #acggcccg     7260     - aaagaagcta cattttgttg ggaagctgga taagttttag gtgcaggacc cc - #aaatgttc     7320     - tgagaccttt ggggccattt attactttgt acaagcccaa taatcctctc tt - #ttctgcca     7380     - agtcctcaac ccagaaatgt aggcttctgt gcaccacacg gcacagccca ct - #gattgctg     7440     - ccaccggctc tgtcttggtc agtgttacca ctgccagcac tcaggctgtg gc - #agatgcca     7500     - gcagctctta ccatcagtca gagtcttcag ggtgtcaagc tgttttcatt tt - #ttaggcaa     7560     - atagaacaaa agccattttg gttcatcctg atcacttgaa tgatagactc aa - #tgccctgt     7620     - gcctggcagg gagcgcttgc agaggtgtcc tagccttaga gggctacttc ag - #tgtctcta     7680     - ctgacagaaa ctcctgtatc tcaaatggat ctcgaagttc tctagtaagg ag - #tcctaagg     7740     - atgacatgta ttgggccact agcagggatt gaaaacattt taaaagaaat cc - #tttttctt     7800     - aggagtaaaa gctggtaaaa ggggtgactt cctggttctg atcaaaacca ga - #ccaaaccc     7860     - tcatttcagc aaagccttgc aagacactcc cttgctcatt tgccatattt ag - #atgtctta     7920     - gtggagtcag agccctgttt ggtatgtgtt tttcatgcta agtctaaatt gt - #cttttcat     7980     - ttcatgatgc attttttctc ttttgtcagg ataacatcat atagcatctt gt - #ttgttttt     8040     - cctaatctct atgaacatat ctatctacct gtaaccgtag ataggtatct ag - #atagatac     8100     - caagctttta agctctgggc cactatgcat cattattggg tctctgcctt aa - #aacacatc     8160     #  8176     - <210> SEQ ID NO 6     <211> LENGTH: 1312     <212> TYPE: DNA     <213> ORGANISM: Mus musculus     <220> FEATURE:     <221> NAME/KEY: CDS     <222> LOCATION: (402)..(1061)     - <400> SEQUENCE: 6     - gcgctgctct gtgagctgga gcacagcgtg cttagagttg gccatattta aa - #atattttc       60     - caataggatc ctgcgtcctt cctccttttc ctccttccct cctccctccc gg - #gtaattta      120     - tttctagctt ccaggcaagg gccacacaag gaaggaaatc cacaggggat ta - #gatgccgg      180     - ggtggtaact ccaccaggct aggttggact ctgcagccaa cttcctatca ga - #tcaccctg      240     - cacctatttc cgacccgacc ggaatgcgac tggcttgagg tccagccctt tc - #gcctgggc      300     - gggagcagag ccgcggaagc tgcttggagt tggatggggg taggaagggg ct - #ggagcggg      360     #ttg cag agg    416tggc ctgggcctaa ggttgggcat a atg gag     #          Met Glu Leu Gln Arg     #         5  1     - aca tcc agc att tca ggg ccg ctg tcg ccg gc - #c tac acc ggg cag gtg      464     Thr Ser Ser Ile Ser Gly Pro Leu Ser Pro Al - #a Tyr Thr Gly Gln Val     #                 20     - cct tac aac tac aac caa ctg gaa gga aga tt - #c aaa cag ctc caa gat      512     Pro Tyr Asn Tyr Asn Gln Leu Glu Gly Arg Ph - #e Lys Gln Leu Gln Asp     #             35     - gag cgt gaa gct gta cag aag aag acc ttc ac - #c aag tgg gtc aat tcc      560     Glu Arg Glu Ala Val Gln Lys Lys Thr Phe Th - #r Lys Trp Val Asn Ser     #         50     - cac ctt gcg aga gtg tcc tgc cga atc aca ga - #c ctg tac acg gac ctt      608     His Leu Ala Arg Val Ser Cys Arg Ile Thr As - #p Leu Tyr Thr Asp Leu     #     65     - cga gat gga cgg atg ctc atc aag cta ctg ga - #g gtc ctc tct gga gag      656     Arg Asp Gly Arg Met Leu Ile Lys Leu Leu Gl - #u Val Leu Ser Gly Glu     # 85     - agg ctg cct aaa ccc act aag gga cgg atg cg - #g atc cac tgt ctg gag      704     Arg Leu Pro Lys Pro Thr Lys Gly Arg Met Ar - #g Ile His Cys Leu Glu     #                100     - aat gtc gac aag gct ctt caa ttc ctg aaa ga - #g cag aga gtc cat ctt      752     Asn Val Asp Lys Ala Leu Gln Phe Leu Lys Gl - #u Gln Arg Val His Leu     #           115     - gag aac atg ggc tcc cat gac att gtg gat gg - #a aac cac cgg ctg aca      800     Glu Asn Met Gly Ser His Asp Ile Val Asp Gl - #y Asn His Arg Leu Thr     #       130     - acg ttg gag cta ctg gaa gtg cgc aga cag ca - #a gag gaa gaa gaa aga      848     Thr Leu Glu Leu Leu Glu Val Arg Arg Gln Gl - #n Glu Glu Glu Glu Arg     #   145     - aag agg cgg cca cct tct ccg gac cca aac ac - #g aag gtt tca gag gag      896     Lys Arg Arg Pro Pro Ser Pro Asp Pro Asn Th - #r Lys Val Ser Glu Glu     150                 1 - #55                 1 - #60                 1 -     #65     - gct gag tcc cag caa tgg gat act tca aaa gg - #a gac caa gtt tcc cag      944     Ala Glu Ser Gln Gln Trp Asp Thr Ser Lys Gl - #y Asp Gln Val Ser Gln     #               180     - aat ggt ttg ccg gct gag cag gga tct cca cg - #g gtt agt tac cgc tct      992     Asn Gly Leu Pro Ala Glu Gln Gly Ser Pro Ar - #g Val Ser Tyr Arg Ser     #           195     - caa acg tac caa aac tac aaa aac ttt aat ag - #c aga cgg aca gcc agt     1040     Gln Thr Tyr Gln Asn Tyr Lys Asn Phe Asn Se - #r Arg Arg Thr Ala Ser     #       210     - gac cat tca tgg tct gga atg tgaagttcac taccatttg - #t caagaaccac     1091     Asp His Ser Trp Ser Gly Met     #   220     - tctgtccaca tcctttgacc ttttggcttc cacgtcaccc agagtgttaa aa - #tttttact     1151     - taattcatag ctgtccttga tttcatattt gtttgcattt aatttatgtt tc - #tttggatc     1211     - ctcattgcct caaagcagca tacttaattt ttgtttattt attgtgagct tt - #ttacttta     1271     # 1312             atca aaattaaatt atagcataat g     - <210> SEQ ID NO 7     <211> LENGTH: 220     <212> TYPE: PRT     <213> ORGANISM: Mus musculus     - <400> SEQUENCE: 7     - Met Glu Leu Gln Arg Thr Ser Ser Ile Ser Gl - #y Pro Leu Ser Pro Ala     #                 15     - Tyr Thr Gly Gln Val Pro Tyr Asn Tyr Asn Gl - #n Leu Glu Gly Arg Phe     #             30     - Lys Gln Leu Gln Asp Glu Arg Glu Ala Val Gl - #n Lys Lys Thr Phe Thr     #         45     - Lys Trp Val Asn Ser His Leu Ala Arg Val Se - #r Cys Arg Ile Thr Asp     #     60     - Leu Tyr Thr Asp Leu Arg Asp Gly Arg Met Le - #u Ile Lys Leu Leu Glu     # 80     - Val Leu Ser Gly Glu Arg Leu Pro Lys Pro Th - #r Lys Gly Arg Met Arg     #                 95     - Ile His Cys Leu Glu Asn Val Asp Lys Ala Le - #u Gln Phe Leu Lys Glu     #           110     - Gln Arg Val His Leu Glu Asn Met Gly Ser Hi - #s Asp Ile Val Asp Gly     #       125     - Asn His Arg Leu Thr Thr Leu Glu Leu Leu Gl - #u Val Arg Arg Gln Gln     #   140     - Glu Glu Glu Glu Arg Lys Arg Arg Pro Pro Se - #r Pro Asp Pro Asn Thr     145                 1 - #50                 1 - #55                 1 -     #60     - Lys Val Ser Glu Glu Ala Glu Ser Gln Gln Tr - #p Asp Thr Ser Lys Gly     #               175     - Asp Gln Val Ser Gln Asn Gly Leu Pro Ala Gl - #u Gln Gly Ser Pro Arg     #           190     - Val Ser Tyr Arg Ser Gln Thr Tyr Gln Asn Ty - #r Lys Asn Phe Asn Ser     #       205     - Arg Arg Thr Ala Ser Asp His Ser Trp Ser Gl - #y Met     #   220     - <210> SEQ ID NO 8     <211> LENGTH: 1964     <212> TYPE: DNA     <213> ORGANISM: Mus musculus     <220> FEATURE:     <221> NAME/KEY: CDS     <222> LOCATION: (322)..(1509)     - <400> SEQUENCE: 8     - ttggaacagt tacttcagtg gaggcagcag aaatgaggct agtccagact ca - #caggaata       60     - gggttccatt ctcaagaaga tgatttaaag taattatcct ttacgcatag tt - #atcatcac      120     - cacaaaaaaa gattccaacc ttttccacag aactattatg atttattttt at - #atgaatgt      180     - atgtatttat tattatatga actcctataa tgatcacctt tacatattca ca - #ttttctta      240     - ataattagtt tagccgcgtc cggaggtccg acagctctgc agctccgagc gc - #gcgactag      300     #att gcc agc cag      351 atg agc cac cag gaa agg     #Ala Ser Glner His Gln Glu Arg Ile     #     10     - agg agg aca aca gcc gaa gtc cca atg cac ag - #a tca act gcc aat caa      399     Arg Arg Thr Thr Ala Glu Val Pro Met His Ar - #g Ser Thr Ala Asn Gln     #                 25     - agc aag agg agc cgg tca cca ttt gcc agc ac - #a cgt cgt cgc tgg gat      447     Ser Lys Arg Ser Arg Ser Pro Phe Ala Ser Th - #r Arg Arg Arg Trp Asp     #             40     - gac agc gag agc tcg gga gcc agc ctg gct gt - #t gag agt gag gat tat      495     Asp Ser Glu Ser Ser Gly Ala Ser Leu Ala Va - #l Glu Ser Glu Asp Tyr     #         55     - tcc agg tgg cgg gat gct gcc gat gct gag ga - #g gct cat gcc gag ggc      543     Ser Arg Trp Arg Asp Ala Ala Asp Ala Glu Gl - #u Ala His Ala Glu Gly     #     70     - cta gcc aga aga ggc cga ggt gag gct gcc ag - #c agc tca gag cca agg      591     Leu Ala Arg Arg Gly Arg Gly Glu Ala Ala Se - #r Ser Ser Glu Pro Arg     # 90     - tat gct gaa gac cag gat gcc agg agt gaa ca - #a gcg aag gca gac aaa      639     Tyr Ala Glu Asp Gln Asp Ala Arg Ser Glu Gl - #n Ala Lys Ala Asp Lys     #                105     - gtg cca aga cgg cgg cga acc atg gca gac cc - #t gac ttc tgg gca tac      687     Val Pro Arg Arg Arg Arg Thr Met Ala Asp Pr - #o Asp Phe Trp Ala Tyr     #           120     - acc gac gat tac tac cga tac tac gag gaa ga - #t tct gac agc gac aaa      735     Thr Asp Asp Tyr Tyr Arg Tyr Tyr Glu Glu As - #p Ser Asp Ser Asp Lys     #       135     - gag tgg atg gct gcc ctg cgc agg aag tac cg - #a agc cga gag caa ccc      783     Glu Trp Met Ala Ala Leu Arg Arg Lys Tyr Ar - #g Ser Arg Glu Gln Pro     #   150     - cag tcc tcc agc gga gaa agc tgg gag ctt ct - #g cca gga aag gaa gaa      831     Gln Ser Ser Ser Gly Glu Ser Trp Glu Leu Le - #u Pro Gly Lys Glu Glu     155                 1 - #60                 1 - #65                 1 -     #70     - ctg gaa cgt cag caa gcc gga gct ggg agc ct - #c gcc agt gct ggc agc      879     Leu Glu Arg Gln Gln Ala Gly Ala Gly Ser Le - #u Ala Ser Ala Gly Ser     #               185     - aat ggc agt ggt tat cct gaa gaa gta caa ga - #c cca tct ctt cag gag      927     Asn Gly Ser Gly Tyr Pro Glu Glu Val Gln As - #p Pro Ser Leu Gln Glu     #           200     - gaa gaa cag gcc tct ctg gaa gaa gga gaa at - #c cct tgg ctt cgc tac      975     Glu Glu Gln Ala Ser Leu Glu Glu Gly Glu Il - #e Pro Trp Leu Arg Tyr     #       215     - aat gag aat gaa agc agc agc gag ggt gat aa - #t gag tct acc cat gag     1023     Asn Glu Asn Glu Ser Ser Ser Glu Gly Asp As - #n Glu Ser Thr His Glu     #   230     - ctc ata cag cct ggg atg ttc atg ctg gat gg - #a aac aac aac ctg gaa     1071     Leu Ile Gln Pro Gly Met Phe Met Leu Asp Gl - #y Asn Asn Asn Leu Glu     235                 2 - #40                 2 - #45                 2 -     #50     - gat gac tcc agc gtg agc gaa gac ctc gaa gt - #g gac tgg agc ctg ttt     1119     Asp Asp Ser Ser Val Ser Glu Asp Leu Glu Va - #l Asp Trp Ser Leu Phe     #               265     - gat ggg ttt gcc gat ggc ttg gga gtg gcc ga - #a gcc atc tcc tac gtg     1167     Asp Gly Phe Ala Asp Gly Leu Gly Val Ala Gl - #u Ala Ile Ser Tyr Val     #           280     - gat cct cag ttc ctc acc tac atg gct ctg ga - #a gag cgt ctg gcc cag     1215     Asp Pro Gln Phe Leu Thr Tyr Met Ala Leu Gl - #u Glu Arg Leu Ala Gln     #       295     - gca atg gag acg gcc ctg gca cac ttg gag tc - #t ctc gcc gtt gat gtc     1263     Ala Met Glu Thr Ala Leu Ala His Leu Glu Se - #r Leu Ala Val Asp Val     #   310     - gaa gtg gcc aac cca cca gca agc aag gag ag - #c att gat gcc ctt cct     1311     Glu Val Ala Asn Pro Pro Ala Ser Lys Glu Se - #r Ile Asp Ala Leu Pro     315                 3 - #20                 3 - #25                 3 -     #30     - gag atc ctg gtc acc gaa gat cat ggt gca gt - #g ggc cag gaa atg tgc     1359     Glu Ile Leu Val Thr Glu Asp His Gly Ala Va - #l Gly Gln Glu Met Cys     #               345     - tgt cct atc tgc tgc agc gaa tat gtg aag gg - #g gag gtg gca act gag     1407     Cys Pro Ile Cys Cys Ser Glu Tyr Val Lys Gl - #y Glu Val Ala Thr Glu     #           360     - cta cca tgc cac cac tat ttc cac aag ccc tg - #c gtg tcc atc tgg ctt     1455     Leu Pro Cys His His Tyr Phe His Lys Pro Cy - #s Val Ser Ile Trp Leu     #       375     - cag aag tct ggc acc tgc cca gtg tgc cgc tg - #c atg ttc cct ccc ccg     1503     Gln Lys Ser Gly Thr Cys Pro Val Cys Arg Cy - #s Met Phe Pro Pro Pro     #   390     - ctc taa aagccaaggc tcgtcgtaac agtcagcctg gttacattcc ct - #gtccgaaa     1559     Leu     395     - cccacaatac tacaggagcc cttgttctaa acttacaatg aaaccagtca gt - #caattaga     1619     - ctaaagttgt tgattccttg tgattatttc catgtgaaaa tggttgtgta ca - #atgacatt     1679     - taaaaaaaat catcctctcg tttagaaggt agaaaggggg aaaggaaact tt - #ctaaatgc     1739     - tgcttgagat tgcagtaaga acatacattt tctaacctga aagttgaaac aa - #atcccact     1799     - tgttctgtag actgtgtctc tcttacctgt tgctgtcagg gttacctatc tg - #ctaaacta     1859     - tgtcggaaag acaaaattac ttttgttgca tgtcatgggt taatgttcct gt - #atttgcag     1919     #                1964aa gttcttcttt tgctttgacc ccgaa     - <210> SEQ ID NO 9     <211> LENGTH: 395     <212> TYPE: PRT     <213> ORGANISM: Mus musculus     - <400> SEQUENCE: 9     - Met Ser His Gln Glu Arg Ile Ala Ser Gln Ar - #g Arg Thr Thr Ala Glu     #                 15     - Val Pro Met His Arg Ser Thr Ala Asn Gln Se - #r Lys Arg Ser Arg Ser     #             30     - Pro Phe Ala Ser Thr Arg Arg Arg Trp Asp As - #p Ser Glu Ser Ser Gly     #         45     - Ala Ser Leu Ala Val Glu Ser Glu Asp Tyr Se - #r Arg Trp Arg Asp Ala     #     60     - Ala Asp Ala Glu Glu Ala His Ala Glu Gly Le - #u Ala Arg Arg Gly Arg     # 80     - Gly Glu Ala Ala Ser Ser Ser Glu Pro Arg Ty - #r Ala Glu Asp Gln Asp     #                 95     - Ala Arg Ser Glu Gln Ala Lys Ala Asp Lys Va - #l Pro Arg Arg Arg Arg     #           110     - Thr Met Ala Asp Pro Asp Phe Trp Ala Tyr Th - #r Asp Asp Tyr Tyr Arg     #       125     - Tyr Tyr Glu Glu Asp Ser Asp Ser Asp Lys Gl - #u Trp Met Ala Ala Leu     #   140     - Arg Arg Lys Tyr Arg Ser Arg Glu Gln Pro Gl - #n Ser Ser Ser Gly Glu     145                 1 - #50                 1 - #55                 1 -     #60     - Ser Trp Glu Leu Leu Pro Gly Lys Glu Glu Le - #u Glu Arg Gln Gln Ala     #               175     - Gly Ala Gly Ser Leu Ala Ser Ala Gly Ser As - #n Gly Ser Gly Tyr Pro     #           190     - Glu Glu Val Gln Asp Pro Ser Leu Gln Glu Gl - #u Glu Gln Ala Ser Leu     #       205     - Glu Glu Gly Glu Ile Pro Trp Leu Arg Tyr As - #n Glu Asn Glu Ser Ser     #   220     - Ser Glu Gly Asp Asn Glu Ser Thr His Glu Le - #u Ile Gln Pro Gly Met     225                 2 - #30                 2 - #35                 2 -     #40     - Phe Met Leu Asp Gly Asn Asn Asn Leu Glu As - #p Asp Ser Ser Val Ser     #               255     - Glu Asp Leu Glu Val Asp Trp Ser Leu Phe As - #p Gly Phe Ala Asp Gly     #           270     - Leu Gly Val Ala Glu Ala Ile Ser Tyr Val As - #p Pro Gln Phe Leu Thr     #       285     - Tyr Met Ala Leu Glu Glu Arg Leu Ala Gln Al - #a Met Glu Thr Ala Leu     #   300     - Ala His Leu Glu Ser Leu Ala Val Asp Val Gl - #u Val Ala Asn Pro Pro     305                 3 - #10                 3 - #15                 3 -     #20     - Ala Ser Lys Glu Ser Ile Asp Ala Leu Pro Gl - #u Ile Leu Val Thr Glu     #               335     - Asp His Gly Ala Val Gly Gln Glu Met Cys Cy - #s Pro Ile Cys Cys Ser     #           350     - Glu Tyr Val Lys Gly Glu Val Ala Thr Glu Le - #u Pro Cys His His Tyr     #       365     - Phe His Lys Pro Cys Val Ser Ile Trp Leu Gl - #n Lys Ser Gly Thr Cys     #   380     - Pro Val Cys Arg Cys Met Phe Pro Pro Pro Le - #u     385                 3 - #90                 3 - #95     - <210> SEQ ID NO 10     <211> LENGTH: 2992     <212> TYPE: DNA     <213> ORGANISM: Mus musculus     - <400> SEQUENCE: 10     - gggcaactga aggcagatga agagccctgc ccctgcccac atgtggaacc tt - #gtgctgtt       60     - cttgccttca ctgttggctg tgcttccgac cactactgcc gagaagaatg gc - #atcgatat      120     - ctacagcctc acggtggact cccgggtctc ttcccgattt gcccatactg tt - #gtcaccag      180     - ccgggtggtc aacagagccg atgctgttca agaagcgacc ttccaagtag ag - #ctacccag      240     - gaaagccttc atcaccaact tctccatgat catcgatggc gtgacctacc ca - #ggggttgt      300     - caaagagaag gccgaagccc agaaacaata cagtgccgcc gtgggcaggg ga - #gagagtgc      360     - tggcatcgtc aagaccactg ggaggcagac agagaagttt gaagtgtcag tc - #aacgtggc      420     - ccctggttcc aagattacct tcgaactcat ataccaggaa ctgctccaaa gg - #cgactggg      480     - aatgtatgag ctactcctca aagtgaggcc tcagcagctg gtgaagcacc tt - #cagatgga      540     - catctacatc tttgagcctc agggtattag catcctggag acagagagca cc - #ctcatgac      600     - cccggagctg gcaaatgccc ttaccacttc acagaacaag accaaggctc at - #atccggtt      660     - caagccgacg ctctcccagc aacagaagtc tcagagtgag caggacacgg tg - #ctgaatgg      720     - ggacttcatc gtccgctatg atgtcaaccg gtctgactct gggggctcca tt - #cagattga      780     - ggaaggctac tttgtgcacc actttgctcc agagaacctt cctacaatgt cc - #aagaatgt      840     - gatctttgtc attgataaaa gcggatctat gtcaggcaag aaaatccagc ag - #acccgaga      900     - agccctagtc aagatcttga aagacctcag cccccaagac cagttcaacc tc - #attgagtt      960     - cagtggggaa gcaaaccaat ggaagcagtc actggtgcaa gcgacagaag ag - #aatttgaa     1020     - caaggctgta aactatgctt ccaggatccg ggctcacgga gggaccaaca tc - #aataatgc     1080     - agtgctgttg gctgtggagc tgctggacag aagcaaccaa gctgagctac tg - #ccctcgaa     1140     - gagcgtctcc cttatcatcc tgctcacgga cggtgacccc actgtgggag aa - #accaaccc     1200     - cacgattatc cagaacaacg tgcgggaagc catcaatggg cagtatagcc tc - #ttctgcct     1260     - ggggttcggc tttgatgtga actatccttt cctggagaag atggcactgg ac - #aatggtgg     1320     - cctggccagg cgcatctatg aggattcaga ctctgcactg cagcttcagg at - #ttctacca     1380     - cgaagtagcc aatccactgc tctcatcagt ggccttcgaa taccccagtg at - #gctgtgga     1440     - ggaagtcact cggtacaagt tccaacacca ctttaagggc tcagagatgg tg - #gtggctgg     1500     - gaagctccag gaccagggtc ctgatgtcct cttagccaaa gtcagtgggc ag - #atgcacat     1560     - gcagaacatc actttccaaa cggaggccag cgtagcccaa caagagaagg ag - #tttaagag     1620     - ccccaagtac atctttcaca actttatgga gagactgtgg gcactgctga ct - #atacagca     1680     - acagctggag cagaggattt cagcgtcagg tgccgaatta gaggccctcg ag - #gcccaagt     1740     - tctgaacttg tcactcaagt acaattttgt cacccctctc acgcacatgg tg - #gtcaccaa     1800     - acctgaaggt caagaacaat tccaagttgc tgagaagcct gtggaagtcg gt - #gatggcat     1860     - gcagagactc cccttagcag ctcaagccca ccccttcagg cctcctgtca ga - #ggatctaa     1920     - actgatgacc gtgctgaaag gaagcaggtc ccagataccc agacgcggtg at - #gccgttag     1980     - ggcatctagg caatacattc ctcccggatt ccccggacct cctggacctc cc - #ggatttcc     2040     - tgcaccccct ggacctcctg gatttcctgc accccctgga cctcctcttg ct - #tctggctc     2100     - tgacttcagc cttcagcctt cctatgaaag gatgctaagc ctgccctccg tt - #gcagcaca     2160     - atatcctgct gacccacatc tggttgtgac ggaaaaaagt aaagaaagca cc - #ataccaga     2220     - ggaatcccca aacccagacc acccccaggt tcctactatt accttgccgc tt - #ccgggatc     2280     - cagtgtggac cagctctgtg tggatatctt acattctgag aagcccatga ag - #ctgttcgt     2340     - agaccccagt cagggtctgg aggtgactgg taagtatgag aatactgggt tc - #tcgtggct     2400     - cgaagtgacc atccagaagc ctcacctgca ggtccatgca acccctgaac ga - #ctggtggt     2460     - gacacgaggc agaaaaaaca ctgaatacaa gtggaagaag acgctgttct ct - #gtgttacc     2520     - tggcttgaag atgaccatga atatgatggg actcctacag ctcagtggcc ca - #gacaaagt     2580     - caccatcggc ctcctgtccc tggatgaccc tcagagagga ctaatgctgc tt - #ttgaatga     2640     - cacccagcac ttctccaaca acgttaaagg ggagcttggt cagttttacc gg - #gacatcgt     2700     - ctgggagcca cccgtcgagc cagataatac aaaacggaca gtcaaagttc aa - #ggagttga     2760     - ctacctggct accagagagc tcaagttgag ttaccaagaa gggttcccag ga - #gcagagat     2820     - ttcctgctgg acagtggaga tatagaactg ttaggagcgc cgctccctgc ca - #tgttgtcc     2880     - tcgtacgcag gcagatgaca ccttatgcca acagggacgc ctgtgaggcc ga - #gaccttga     2940     - tgggaagagg atgctccctt gttacaaata aagaagggca gtgtgaaccc ga - #     2992     - <210> SEQ ID NO 11     <211> LENGTH: 1177     <212> TYPE: DNA     <213> ORGANISM: Mus musculus     <220> FEATURE:     <223> OTHER INFORMATION: For all n's in this - # sequence, n=(a or g or           c or t)     - <400> SEQUENCE: 11     - ggtggccaag agcagttcac ctgctctggg gcaagccttg cttgtgtttt ag - #tgagtcag       60     - ggcctcccca ggcagtaaga tgttgagtgt ggaggcccag gccgctgacc tg - #cagccctg      120     - tcccccacag gcaggctgca tgctcttccc ccacatttct ccttgcgagg tg - #cgcgtgct      180     - catgctcctg tactcgtcta agaagaagat cttcatgggc ctcatcccct ac - #gaccagag      240     - cggnttcgtc aacgccatac gacaggtcat caccacccgc aaacaggtgt gc - #cagctgag      300     - ggtagnctgc tcctgctcct acccttggta gacccactgn ctcccactgg tg - #tggaatgt      360     - ggcatcaagg ctgagtcggc gnctggggag gagctgtgac gangcagtgc ca - #tacccaaa      420     - tgggctcgag ggaaacntag ctttataggc ttcagagggg cagaactaga gg - #gtggggcc      480     - tgggtgtaga ggcagggcag gagtggggtg gcaggtttgg caagaggccc ag - #agtctctg      540     - gagggtcaca gtgttgatga catctttctn agaancctgc tactngctta gn - #cagctgtg      600     - gtcctctctn ccacctgggg gatacctggc nacaggcngt gggcnncggg gg - #tgaanact      660     - ctggacctgt tnagantgtc aacaacaaat tcttgacatg gagtggtgtc at - #ggagtggn      720     - aggaggtgan ctgccgggga ctgtgtggac tgttgnccct aagctgccct cc - #cctgaagt      780     - gccttctcgc tctgccccaa aacccagacc tgagcccaac agccggtcca ag - #aggtggct      840     - gccatcccac gtctatgtga accaagggga gatcctgtga ttccgggtac cc - #ccgggtgg      900     - ccccattgac agtgccgccc ccctggggga ggacttctga ctgatacctc ct - #gtcttgtg      960     - tggcaggaga acagaccagt ggcctcggag gctcttcatg cagctcattc cc - #cagcagtt     1020     - gctggtgagg ggtcagggga ttccaggctg ggggtgggcc aaagaccctg tg - #gtgggctg     1080     - gttcagaggc ctgcctggct tccccagcaa gctagggttc cataaagaag cc - #ctcggcct     1140     #    1177          cgtg ccactgttcc ggaattc     - <210> SEQ ID NO 12     <211> LENGTH: 2998     <212> TYPE: DNA     <213> ORGANISM: Mus musculus     <220> FEATURE:     <223> OTHER INFORMATION: For all n's in this - # sequence, n=(a or g or           c or t)     - <400> SEQUENCE: 12     - ggcacgagct taactgtgct aacttctgtg atgatcatgt gtgatgagta tg - #tgctctca       60     - tttgatttgt gggaaaaaac aaaacaaaaa aatccgaagg acacaaagag ga - #ctaatctt      120     - aaaccagata tctagtagtc accaaagcca cactttgaat tcgaaaagct ta - #gcactgta      180     - gcttagctca tgctatcttt taaagagaga atttaattat ttaatatatg ga - #aggacatt      240     - aggctagtgt gtctggcaca tggtataaac tcaataaatg gtggacgtta tc - #agtgctac      300     - tataatgagt ttaataattt ggtttcatct cctttaatca gaccagtgtt ca - #ctactagc      360     - tgggtctctg gaataggcac agatatattc atctggagtg tcacacatac tc - #tgtgcgcg      420     - aaagagttca gaatagccct tcaataagcc aattactctt gctgtcatcc tt - #atttctta      480     - actttccctt agcgttgctt ttatgtatca aacttttctt ccttatttta cg - #taatactt      540     - ttaatgacaa ctttctagaa ataagaacta taccctaaaa gattgaaata tt - #cttagttt      600     - tctttatcta catcagaaat tgtttagctg atacaacata cttatattgt tt - #aaggaatt      660     - ctgtttaata ccttggtatt tataattttc ataagtttat ttgtattaat ag - #gaactctt      720     - acaaagaatg tatagaaaat aagccccatc atttgtcagt gtgacaattt tc - #ccagtgtt      780     - taaattgttt aagctgtttg tacccctata taagctctgt tccttctttg gc - #cctttccc      840     - ccttagccta aatctccatt ttgcctgacg atctcttccc tgacaaaatg cc - #tgcttctg      900     - cgcactgagt cacagtctac taaaatgcat tccattgtgc ccatgtccct ct - #taatgtga      960     - tgaccccaga catgaccagg gcagagcaca gagggagcat cactttcttt ga - #ccagagca     1020     - tctatttcca gcaatgcagc ctaaggtcac attagcattt ttggcagcaa aa - #tacaccct     1080     - tggctcatgc tgttatgctg tcaaccaaat cctccatgac tttttcacat ga - #actcccat     1140     - taaataaggc ttcccacatc cggtacgaat atagacagta atgtgcagtc tg - #gtgaagtt     1200     - atttacataa gttcctatta aacatcagct aatctatatt tattatttta ga - #atattgag     1260     - acagatttct attcccagct atatagatat ggttttagaa tactttatta tt - #attttttt     1320     - aatgtgtctt ctctgaaccc gataagaaca tagtcccaga caatctttaa gt - #tcagagtc     1380     - ttacagtttg tatagagacc tagaggctag ctatatttct ttagacatca ac - #acatcatc     1440     - agataggatc cacccaaggg ccttacaaat cctgtatact gaaatgcctt tt - #cctgacga     1500     - tattctggag actgttaagt gaatgcgcag atctgaaccg agccgagcct gt - #agtgggga     1560     - agagctaaag catggcagtt gtcttcatca atgatggagt ctttcattat gt - #tgtctcaa     1620     - aagacacatg cttcagccct gggtctcaaa actctcatgc ttcggccctg gg - #tctcacac     1680     - tcctggcttc ccgagtggtc atagctaaga ccttctcaca ctaaatccca gg - #atgagctc     1740     - atgttgatgt tcctgcttgc ttctctgaaa ttggcagttc tcgtgggaaa aa - #aaatctac     1800     - ttatacttgt gtgcttcata aagcaactcg gtagcagggc ttaggggtgc tt - #cgagtgtg     1860     - gcagtgatag agaagaccga taaagcgaaa tctatgatat ctcatacatc at - #tttaatta     1920     - tttaaattac ttttgttagt acacaaaagt attttgttag tacaccctgt tt - #atctatgt     1980     - gtatactcta cctttcgcat acactgactt catttctttt tctcctcacc ca - #tcctgatg     2040     - agctgctctc ctcccagaca agctctggca gttttaaagt cacgtgtgta tc - #ttttaact     2100     - ctagcttctg cctattagac aaaacaagat acttgtcttt ctccccatct cc - #ctcctttt     2160     - gtttaattct cctccagccc tacatggatc ccccttgacc tcgtgtcata ta - #tctaaatc     2220     - tgtataaata aagagatgat ttaatctacg ttctatgtac aaaagagaat at - #aaatgctc     2280     - gtctttctga atctgtctta tttggtttca cacaatatct gctctctttt ac - #cgcaaatg     2340     - gtatcatctc gttcccttta cacgttgaag aaaatttcat tttgtgtgtg tg - #tgtgtgtg     2400     - tgtgtgtgtg aactatatat ttttacgcta tctggtgagg aacatcaagg cc - #aagatatg     2460     - gatcttggct attgtaaaga gtgtagtaag aaacacaacc gtataatcat ct - #ctgttgca     2520     - tgctggcatg ctggctacaa tcctcacctg tgtacccaga gtgagagctg ga - #ccacatgg     2580     - taatgcaacc tgtagttatt atttaatgtg tacttcttgt ttaatgttta aa - #gatactac     2640     - ttattttaat gttatgtgta tggatgtttt atctatgtgt ttgtctgtat at - #agtgggca     2700     - cgtactggtc tcagagccag aggaaggcat cagagtccct ggggttggaa tt - #aaagatgt     2760     - ttgtgagtac ctgcgtgtat cctggacttc aaacccgggt cttcttcaag ag - #cagccagt     2820     - gctcttaacc actgaggatc tctccagcct catcgctgat ttaggaagga ct - #tttactga     2880     - tttggagtag ctgtaggcaa tgcagtctat gacgatttcc ttttagcagt tc - #ttgtttgt     2940     - tttcttaatg atagccatac tgattgctga gatttacagc agcactagca ag - #ctggaa     2998     - <210> SEQ ID NO 13     <211> LENGTH: 1121     <212> TYPE: DNA     <213> ORGANISM: Mus musculus     <220> FEATURE:     <223> OTHER INFORMATION: For all n's in this - # sequence, n=(a or g or           c or t)     - <400> SEQUENCE: 13     - ctcgagtttt tttttttttt ttttggagaa gggnaacatt tattcattca ac - #aaatnttg       60     - atgacctgat ggggnagata actgagctag tcagcgcgta ggtagcaaac at - #aaggntat      120     - agtaccccag ntaatggtct ncccacatgt cactgaagga gtgtcagttc tc - #agcatttt      180     - acctttaatt ttaattttta cctctaaatg cgctttagga ggctacccac ag - #ttgatgac      240     - aaacagtgta gccaggcatg ccagaactgt taccagcaga acttttggcc ga - #ctgtagct      300     - ggcagtgttc tcagtagtgc agttcatgcc tggtgggtgt aactagggta ca - #acgaagtc      360     - actttgaact cttttgctaa ctaaataagc caaataaaca aatcatgaaa ta - #ctgattag      420     - caatgcaata tttcatggca tgggaagagc ttcgacttct ccatcggtga ca - #aggagcag      480     - cttctggaag gaaggtctgg agaaaacaac tgacggggag ctccgaggag cc - #ctgaacac      540     - gtcactcaac agcactggcg ttgacacagc tgctgtggtc cagcagtcac tc - #agtggaga      600     - gtgccaaagg gtgggcagac agncagncct acttcttcat ctccaggatg gc - #acttccag      660     - gcccacggtt cttagcacta cagatgttgc agtattgtgc aggagcattc at - #gctcggca      720     - taggcaggca ctccttgtgg aacatgtgcc ggcagtggaa gaccaccacg ct - #gaagggct      780     - tcnctgcatc tgttgggagg atgggagaaa ggcatgattc acagatattc tc - #ttcatcaa      840     - ccagaacgcc tttcatttgg gttcggngca ttttttcaca caccaacgac aa - #tgagtcag      900     - ctacgaggat tttcttgcag ccttcccgaa gcagaatctt caagttataa tc - #ttgcagaa      960     - ttttaaccaa ggaatctctc aaattgggaa tctccattcc ttccttaatt cg - #gtggataa     1020     - gtagaatcgg gtccacatgt gtgccaatgt tgttcaacaa gccagtgata aa - #tggtggtt     1080     # 1121             aatc agatcttctc gtgccgaatt c     - <210> SEQ ID NO 14     <211> LENGTH: 779     <212> TYPE: DNA     <213> ORGANISM: Mus musculus     - <400> SEQUENCE: 14     - ctcgagagat gccccacagt ccctcaggac ccgagtcagg taatctgcct tt - #ggccttag       60     - tgacctcctt ttctgggcga gtataccatc cactttcctc cctgacaggc ag - #ttcagtaa      120     - cccaaccctt tcattcctcc ttcagttgtc aaagacaact taacatccaa ga - #ctaacaag      180     - caagatgact caggagcatg gnctctgggt tcccctggca ccatgcatgg tg - #atgctagt      240     - taaggctgac ttagctctta gcaaccttgg ttgggatagc ttaagctcat ct - #ccactttc      300     - ctaccaaaca gaaaagaatt tgagtcctct tgctatgagg ctctcgctcc ca - #tctcaggc      360     - gagcttcctg cccctcaccc aagcttggga ggtagagtta tggagagggc aa - #ggaagcag      420     - gactggaaag atagacttat ggatccacca ctcataaagt cacaaagtcc cc - #tcacacct      480     - gctagactta gactctaaat cattacgttg tcaccaacag aggtgactcc tc - #aaccacaa      540     - gagcctgtag tgagcttcaa gagagaagag gacaagncag acctggactg ca - #tgaccttg      600     - cacctgtgat gaagtcacag caataggtga tgctcaaaaa gccccaataa aa - #tgcaagac      660     - agncaaacag aagccctgtc tgtccccatt ggtgggtaat gtagctgatg tg - #gctggttc      720     - tccttccttg acttcaccct gactatggga attgtccttc agtgcctcgt gc - #cgaattc      779     - <210> SEQ ID NO 15     <211> LENGTH: 981     <212> TYPE: DNA     <213> ORGANISM: Mus musculus     <220> FEATURE:     <223> OTHER INFORMATION: For all n's in this - # sequence, n=(a or g or           c or t)     - <400> SEQUENCE: 15     - ctcgagaggt gaaggcagaa gtatcacaag ttcaagttca aggncagcct gg - #gcttcaca       60     - agacccaaaa aaataaatat gaggncagtc caggctggga ctcaggtcac tg - #ctgtgctg      120     - agccatcgtc agagaagttt cttctttnnt tttgatagga gctaacacag cg - #acccacan      180     - ctggacagnc tgcagtgagt gagtgagtaa gtgacctaaa agtgatgtct tc - #attaatct      240     - cccctcccca ggcntcaggg agctctgagg aagaggaggc agaaagatgg tg - #agagccag      300     - cagggatgga ggacaccaag gaagcagtgt cttccgacac aacaggactg gc - #atttagga      360     - agtcacagag gctgtggctg cccagggcct gcacggtcca agctggctga ga - #ttccagtg      420     - ctgagagaga caattcaaca cggnctccca cccctagnca agaagttatc tc - #caactgat      480     - atccacttgc aaaggaaaaa attagggggn tagagagatg gctcagtggn ta - #agagcact      540     - gacttanaaa atagaaatng canattngnt nngangttng cnaaatngct ga - #gaaatggc      600     - caattggctg gaaaacttgc aacattgcct ggagaactgc caaattgcct gg - #agagctgc      660     - caaattggcc tggagagctg cctacatggc ctggagagct gcccacatgg cc - #tggagaac      720     - tggctacatg tcctggagag ctgccaacat gtcctggaga tctgcctaca tg - #gcctggag      780     - aactgcctac atgacctgga gagctggcca catggcctgg agagctggct ac - #atgacctg      840     - gagagctgnc tacatggcct ggagagctgg ctacatggcc tggagagctg gc - #tacatggc      900     - ctggagagct ggctacatgg cctggagagc tggctacatg gcctggagag cc - #tcccagca      960     #                 981tt c     - <210> SEQ ID NO 16     <211> LENGTH: 685     <212> TYPE: DNA     <213> ORGANISM: Mus musculus     <220> FEATURE:     <223> OTHER INFORMATION: For all n's in this - # sequence, n=(a or g or           c or t)     - <400> SEQUENCE: 16     - ctcgagatgc attaaagctt tgntgcagaa ggatccgagt gtgtcctgtg tg - #tgtgtcct       60     - cactggcgag accctttatc acacagggac accccttagg ttggagtttt cc - #ttgtaatg      120     - tccactatac gtctgcttta tacaataata ttgnttaaat ttgnctctat ca - #tgaaatac      180     - ctcactttcc ttatctgtat tgattgaaag ttttggtgga tgtaatagtt tg - #ggcttgga      240     - tctgaagtct tttagagttt attggacatg tgcctngatt cattggnttn aa - #aatcntcc      300     - acnacttggg ggtgtaaagg ttacccacnc nattantgga ggttcttctg ag - #ttccagag      360     - anaangantg agccaccngg aattctccct aaacacactt tgatcatttc ct - #gcctaacc      420     - ctgcagagga aatattaata ccctgtagta ccaaaggaaa caaataagaa gg - #aagactgn      480     - tctctcatgt ctggaggaag tttggtgaag gagtcttctg tttgctcaca ta - #ggagagat      540     - ctaatacagc cactatccat aattaaaaat ctctgtgaga gaggcatgac ga - #ggttctcc      600     - cagtctgtca agggatgtga atatgtgttn ccctgtcatc ctgtcatgaa gc - #ctctcttt      660     #              685 gccg aattc     - <210> SEQ ID NO 17     <211> LENGTH: 471     <212> TYPE: DNA     <213> ORGANISM: Mus musculus     <220> FEATURE:     <223> OTHER INFORMATION: For all n's in this - # sequence, n=(a or g or           c or t)     - <400> SEQUENCE: 17     - gaattcngcn ttggggtaca tggaccngga gagcttggnt acatggcctg ga - #gagctggn       60     - tacatggccc ggngagctgg tttnataaac ctggggangt tgggttnaat gg - #ccccgggg      120     - angtnggttn aatanaccng gggaggtgtc tgaaaanagt ggncacgtac tg - #ttctcaga      180     - cccagnggaa gncatcagag tcccctgggg ttggaattaa agatgtttgt ga - #gtcnctgc      240     - gtgtatcctg gacttcaaac ccgggtcttc ttcaagagca gccagtgctc tt - #aaccactg      300     - agggatctct ccagcctcat cgctgattta ggaaggactt ttactgattt gg - #agtanctg      360     - tagccaatnc agtctatgac gatttccttt tagcagttct tgtttgtttt ct - #taatgata      420     #            471ctgagat ttacagcagc actagcaagc tggaactcga g     - <210> SEQ ID NO 18     <211> LENGTH: 467     <212> TYPE: DNA     <213> ORGANISM: Mus musculus     <220> FEATURE:     <223> OTHER INFORMATION: For all n's in this - # sequence, n=(a or g or           c or t)     - <400> SEQUENCE: 18     - ctcgagnttt tttttttttt tttttttttt tttttttttt tttttttttt tt - #tttttttt       60     - tttttnnnnn aanaaanttt taaagttttt ttttttttat naaaannttt cc - #aagggggg      120     - gangggttag aaganagcca nagcctggnc ccccctgcca gaaaaaacca ga - #ggggggtt      180     - gatgtcccca agtccagttg tcaccctgaa gaagttcccc acgatttccc tg - #gtggcccc      240     - ccgggagtac gtccagagtg tcaccctttc catttgggag ctgtgggaag gg - #ngtgggnt      300     - ccctcccagn ggggccccaa acccttctcc tgaacagntc ctgatttctg ac - #catctttc      360     - caattccacg gattcaaaga gcatgaccct aggtaagcaa gccaggtcaa ga - #gcattgct      420     #               467aagg gtccctcctg gcctcgtgcc gaattcc     __________________________________________________________________________ 

What is claimed is:
 1. An isolated nucleic acid encoding an early liver developmental protein, said nucleic acid having a nucleotide sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17 and SEQ ID NO:18.
 2. An isolated nucleic acid encoding a protein having an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:7 and SEQ ID NO:9. 